Methods for isolating molecular mimetics of unique Neisseria meningitidis serogroup B epitopes

ABSTRACT

Novel bactericidal antibodies against  Neisseria meningitidis  serogroup B (“MenB”) are disclosed. The antibodies either do not cross-react or minimally cross-react with host tissue polysialic acid and hence pose minimal risk of autoimmune activity. The antibodies are used to identify molecular mimetics of unique epitopes found on MenB or  E. coli  K1. Examples of such peptide mimetics are described that elicit serum antibody capable of activating complement-mediated bacteriolysis of MenB. Vaccine compositions containing such mimetics can be used to prevent MenB or  E. coli  K1 disease without the risk of evoking autoantibody.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.09/910,552, filed Jul. 23, 2001, now U.S. Pat. No. 6,642,354, which is adivisional of U.S. patent application Ser. No. 09/494,822, filed Jan.31, 2000, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 08/925,002, filed Aug. 27, 1997, now U.S. Pat. No.6,048,527, from which applications priority is claimed pursuant to 35U.S.C. §120; and is related to provisional patent application Ser. No.60/025,799, filed Aug. 27, 1996, from which application priority isclaimed under 35 U.S.C. §119(e)(1) and which applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention pertains generally to bacterial pathogens. Inparticular, the invention relates to antibodies that elicit functionalactivity against Neisseria meningitidis serogroup B and also lackautoimmune activity, methods of obtaining and using the same, as well asmolecular mimetics identified using the antibodies.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a causative agent of bacterial meningitis andsepsis. Meningococci are divided into serological groups based on theimmunological characteristics of capsular and cell wall antigens.Currently recognized serogroups include A, B, C, D, W-135, X, Y, Z and29E. The polysaccharides responsible for the serogroup specificity havebeen purified from several of these groups, including A, B, C, D, W-135and Y.

N. meningitidis serogroup B (“MenB”) accounts for approximately 50percent of bacterial meningitis in infants and children residing in theU.S. and Europe. The organism also causes fatal sepsis in young adults.In adolescents, experimental MenB vaccines consisting of outer membraneprotein (OMP) vesicles have been found to be approximately 50%protective. However, no protection has been observed in vaccinatedinfants and children, the age groups at greatest risk of disease.Additionally, OMP vaccines are serotype- and subtype-specific, and thedominant MenB strains are subject to both geographic and temporalvariation, limiting the usefulness of such vaccines.

Effective capsular polysaccharide-based vaccines have been developedagainst meningococcal disease caused by serogroups A, C, Y and W135.However, similar attempts to develop a MenB polysaccharide vaccine havefailed due to the poor immunogenicity of the capsular MenBpolysaccharide (termed “MenB PS” herein). MenB PS is a homopolymer of(N-acetyl (α2→8) neuraminic acid. Escherichia coli K1 has the identicalcapsular polysaccharide. Antibodies elicited by MenB PS cross-react withhost polysialic acid (PSA). PSA is abundantly expressed in fetal andnewborn tissue, especially on neural cell adhesion molecules (“NCAMs”)found in brain tissue. PSA is also found to a lesser extent in adulttissues including in kidney, heart and the olfactory nerve. Thus, mostanti-MenB PS antibodies are also autoantibodies. Such antibodiestherefore have the potential to adversely affect fetal development, orto lead to autoimmune disease.

MenB PS derivatives have been prepared in an attempt to circumvent thepoor immunogenicity of MenB PS. For example, C₃–C₈ N-acyl-substitutedMenB PS derivatives have been described. See, EP Publication No. 504,202B, to Jennings et al. Similarly, U.S. Pat. No. 4,727,136 to Jennings etal. describes an N-propionylated MenB PS molecule, termed “NPr-MenB PS”herein. Mice immunized with NPr-MenB PS glycoconjugates were reported toelicit high titers of IgG antibodies. Jennings et al. (1986) J. Immunol.137:1708. In rabbits, two distinct populations of antibodies,purportedly associated with two different epitopes, one shared by nativeMenB PS and one unshared, were produced using the derivative.Bactericidal activity was found in the antibody population that did notcross react with MenB PS. Jennings et al. (1987) J. Exp. Med. 155:1207.The identity of the bacterial surface epitope(s) reacting with theprotective antibodies elicited by this conjugate remains unknown.

Peptides can serve as mimics of polysaccharides by binding topolysaccharide-specific antibodies as well as to other polysaccharidebinding proteins. For example, concanavalin A (Con A), which binds tooligosaccharides bearing terminal alpha-linked mannose or glucoseresidues, has been used to select peptide mimetics from random librariesof bacterial phage bearing short peptide sequences at the amino-terminusof the pIII coat protein. Olderberg et al. (1992) Proc. Natl. Acad. Sci.USA 89:5393, Scott et al. (1992) Proc. Natl. Acad. Sci. USA 89:5398.Similarly, monoclonal antibodies have identified peptide mimetics of acarbohydrate present on the surface of adinocarcinona cells from a phagelibrary Hoess at al. (1993) Gene 128:43.

Peptides can also elicit polysaccharide-specific antibodies. Forexample, Westerink et al. (1988) Infect. Immun. 56:1120, used amonoclonal antibody to the N. meningitidis serogroup C (“MenC”) capsularploysacceride to elicit an anti-idiotype antibody. Mice immuniced withthe anti-idiotype antibody were protected against infection with alethal dose of MenC bacteria. These experimenters subsequentlydemonstrated that a peptide fragment of a MenC anti-idiotype antibodyeliciten serum anti-MenC antibodies and protected animals frombacteremia and death after lethal challenge with MenC bacteria.Wescerink et al. (1995) Proc. Natl. Acad. Sci USA 92:4021.

However, to date, no such approach has been taken with respect to MenBvaccine development. It is readily apparent that the production of asafe and effective vaccine against MenB would be particularly desirable.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of functionally activeantibodies directed against MenB PS derivatives, wherein the antibodiesdo not cross-react, on are minimally cross-reactive, with host tissuesas determined using the assays described herein. These antibodiestherefore pose minimal risk of evoking autoimmune disease and are termed“non-autoreactive” herein. Assays used herein to determineautoreactivity include binding assays against a neuroblastoma cell lineexpressing long chain polysiatic acid residues on the cell surface.Specifically, antibodies that are negative in these assays areconsidered to lack autoreactivity. The non-autoreactive antibodies areparticularly useful for identifying molecular mimetics of unique MenB PSepitopes that can be used in vaccine compositions. Furthermore, theantibodies humanized versions of the antibodies fragments and functionalequivalents thereof, will also and use in passive immunization against,and/or to a adjunct to therapy for, MenB and E. coli K1 disease Sincesuch molecules do not bind to polysialic acid in tissues as determinedby the autoreactivity assays described herein, they provide a safe andefficacious method for the treatment and/or prevention of MenB and E.coli K1 disease.

Accordingly, in one embodiment, the subject invention relates toantibodies directed against MenB PS derivatives, wherein the antibodiesare not autoreactive with host tissue. Such antibodies may further becharacterized as being capable of eliciting functional activity againstMenB bacteria. One particular group of such antibodies is alsocharacterized as non cross-reactive with Neisseria meningitidisserogroup B capsular polysaccharide (NAc-MenB PS) in an ELISA. However,these antibodies are anti-capsular in that they can bind to the cellsurface of a Group B encapsulated bacteria, but not tocapsular-deficient mutants.

Another embodiment of the invention relates to monoclonal antibodiesdirected against MenB PS derivatives, and hybridomas producing thosemonoclonal antibodies.

Other embodiments of the invention relate to unique Neisseriameningitidis serogroup B epitopes that are capable of being bound by theantibody molecules of the present invention.

Still further embodiments of the subject invention are related tomethods for isolating molecular mimetics of unique epitopes of MenB PSand molecular mimetics identified using the methods. The methodscomprise:

(a) providing a population of molecules including a putative molecularmimetic of a unique epitope of MenB PS;

(b) contacting the population of molecules with the antibodies describedabove under conditions that allow immunological binding between theantibody and the molecular mimetic, if present, to provide a complex;and

(c) separating the complexes from non-bound molecules.

In another embodiment, the subject invention is directed to a vaccinecomposition comprising a unique epitope of MenB in combination with apharmaceutically acceptable excipient.

In yet another embodiment, the invention is directed to a vaccinecomposition comprising a molecular mimetic of a unique epitope of MenBin combination with a pharmaceutically acceptable excipient.

In still a further embodiment, the invention is directed to a vaccinecomposition comprising an anti-idiotypic antibody molecular mimetic of aunique epitope of MenB in combination with a pharmaceutically acceptableexcipient.

In yet further embodiments, the invention relates to pharmaceuticalcompositions comprising the antibodies described above.

In another embodiment, the subject invention is directed to a method fortreating or preventing MenB and/or E. coli K1 disease in a mammaliansubject comprising administering an effective amount of the abovepharmaceutical compositions to the subject.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1D depict dose-response binding activity of threerepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-5,SEAM-16 and SEAM-18, respectively), to solid phase NPr-MenB PS asdetermined by ELISA. Data shown are for the antibodies diluted in buffer( ), or in buffer containing 25 μg/ml of soluble NPr-MenB PS (∘).Different ranges for the X axis in the data are used, wherein monoclonalantibodies SEAM-3, SEAM-16, and SEAM-18 are shown at 0.0001 to 1 μg/ml,and monoclonal antibody SEAM-5 is shown at 0.1 to 100 μg/ml.

FIG. 2 depicts the inhibition of binding of four representativeanti-NPr-MenB PS monoclonal antibodies (SEAM-2, SEAM-3, SEAM-16 andSEAM-18) to solid phase NPr-MenB PS by either 25 μg/ml of soluble highmolecular weight (HMW) NPr-MenB PS inhibitor (▪), or 25 μg/ml of lowmolecular weight (LMW) NPr-MenB oligosaccharide inhibitor having anaverage degree of polymerization of 3.8 monomers (□), as determined byELISA.

FIG. 3 depicts the binding of five representative anti-NPr-MenB PSmonoclonal antibodies (SEAM-12, SEAM-16, SEAM-18, SEAM-2, and SEAM-3) tosolid phase NAc-MenB PS as determined by ELISA. Three of the antibodies,SEAM-12, SEAM-16 and SEAM-18, showed significant binding when tested at0.5 and/or 5 μg/ml of antibody. Two other antibodies, SEAM-2 and SEAM-3,were negative when tested at 5-fold higher concentrations (25 μg/ml ofantibody).

FIGS. 4A–4G depict the cross-reactivity of control antibodies andrepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-18,SEAM-9, SEAM-10, and SEAM-7) with encapsulated and non-encapsulatedwhole MenB bacteria as determined by indirect fluorescence flowcytometry. The capsule contains NAc-MenB PS.

FIGS. 5A–5D depict the complement-mediated bactericidal activity of fourrepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-5,SEAM-12, and SEAM-18, respectively) when tested against the MenB teststrain 8047. Results are shown from experiments with three differentcomplement sources: infant rabbit complement I (▴); infant rabbitcomplement II ( ); and human complement (o).

FIGS. 6A–6I depict the cross-reactivity of three control antibodies andfour representative anti-NPr-MenB PS monoclonal antibodies (SEAM-5,SEAM-35, SEAM-12, and SEAM-7) with polysialic acid antigens displayed onthe surface of the human neuroblastoma cell line CHP-134 as determinedby indirect fluorescence flow cytometry.

FIGS. 7A–7B depict the amino acid sequences of 67 unique peptide mimeticsequences (SEQ ID NOs. 1–67) selected by SEAM monoclonal antibodies fromphage display peptide libraries.

FIGS. 8A and 8B depict the ELISA binding activity of sevenrepresentative SEAM monoclonal antibodies (SEAM-2, SEAM-3, SEAM-5,SEAM-7, SEAM-12, SEAM-16, and SEAM-18) to two peptides containingpeptide mimetic sequences selected by SEAM monoclonal antibodies. (InFIG. 8A, “Pep 4” is Lauryl-GLY-GLY-[SEQ ID NO. 4]-Amide, and in FIG. 8B,“Pep 8” is Lauryl-GLY-GLY-[SEQ ID NO. 8]-Amide). Each peptide contains acarboxyl terminal amide and a Lauryl-Gly-Gly at the amino terminal endin order to facilitate binding of the peptide to the microtiter plate.

FIG. 9 depicts the antibody binding activity of pooled (four mice perpool) immune and unimmunized (CTL) sera from CD1 mice as measured by anELISA with peptide Pep 8 as the solid phase antigen. The immune serawere from mice immunized with 5 μg or 50 μg of mimetic peptidescomplexed to the capsule-deficient Neisseria meningitidis Strain M7outer membrane protein vesicles. The peptides included Pep 5(Lauryl-GLY-GLY-[SEQ ID NO. 5]-Amide), Pep 8 (Lauryl-GLY-GLY-[SEQ ID NO.8]-Amide), or a mixture of nine peptides Pep 1 through Pep 9 (Pep 1,Lauryl-GLY-GLY-[SEQ ID NO. 1]-Amide; Pep 2, Lauryl-GLY-GLY-[SEQ ID NO.2]-Amide; Pep 3, Lauryl-GLY-GLY-[SEQ ID NO. 3]-Amide; Pep 4,Lauryl-GLY-GLY-[SEQ ID NO. 4]-Amide; Pep 5, Lauryl-GLY-GLY-[SEQ ID NO.5]-Amide; Pep 6, Lauryl-GLY-GLY-[SEQ ID NO. 6]-Amide; Pep 7,Lauryl-GLY-GLY-[SEQ ID NO. 7]-Amide; Pep 8, Lauryl-GLY-GLY-[SEQ ID NO.8]-Amide; and Pep 9, Lauryl-GLY-GLY-[SEQ ID NO. 9]-Amide). Binding iscompared between sera diluted in buffer (□), buffer containing solublePep 8 (Acetyl-[SE ID NO. 8]-Amide) (▪), or buffer containing a solubleirrelevant peptide R1 (Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO.68)) (cross-hatched bars).

FIG. 10 depicts the antibody binding activity of pooled (four mice perpool) immune and unimmunized control sera from CD1 mice as measured byan ELISA with NPr-MenB PS as the solid phase antigen. The mice wereimmunized with the peptide immunogens as described above in FIG. 9.

FIG. 11 depicts the antibody binding activity of pooled (four mice perpool) immune and unimmunized control sera from CD1 mice as measured byan ELISA with NAc-MenB PS as the solid phase antigen. The mice wereimmunized with the peptide immunogens as described above in FIG. 9. TheSEAM-30 antibody, with known autoantibody activity, served as thepositive control.

FIGS. 12A–12B depict the percent survival of bacteria incubated withvarious dilutions of test sera and human complement. The data shown arefrom testing pooled sera (four mice per pool) from CD1 mice immunizedwith 5 μg (FIG. 11A) or 50 μg (FIG. 11B) of mimetic peptide Pep 8(Lauryl-GLY-GLY-[SEQ ID NO. 8]-Amide) complexed to capsular-deficientNeisseria meningitidis Strain M7 outer membrane protein vesicles. Thesera were diluted in buffer, or in buffer containing Pep 8 inhibitor(100 μg/ml). The source of complement was human agammaglobulinemia andthe bacterial test strain was 8047.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of immunology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); and Handbook of Experimental Immunology, Vols. I–IV (D. M. Weirand C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, a “MenB PS derivative” refers to a molecule obtained bythe chemical modification of the native capsular polysaccharide of MenB.Such MenB PS derivatives include, but are not limited to, MenB PSmolecules which have been modified by the substitution of sialic acidresidue N-acetyl groups of the native molecule with appropriate acylgroups, such as C₃–C₈, and higher, acyl groups wherein the term “acylgroup” encompasses any acylated linear, branched, aliphatic or aromaticmolecule. A particularly preferred MenB PS derivative for use hereincomprises the substitution of N-propionyl groups for N-acetyl groups ofnative MenB PS (termed “NPr-MenB PS” herein). Methods for synthesizingN-acyl-substituted MenB PS derivatives, including NPr-MenB PS, are knownin the art and described in e.g., U.S. Pat. No. 4,727,136 to Jennings etal. and EP Publication No. 504,202 B, also to Jennings et al.

“Molecular mimetics” of MenB PS, or derivatives of MenB PS are moleculesthat functionally mimic at least one “unique” epitope expressed on aMenB bacteria. A “unique epitope” is an epitope capable of eliciting theformation of functionally active (e.g., opsonic and/orcomplement-mediated bactericidal) anti-MenB antibodies that either arenot cross-reactive with polysialic acid in host tissue and hence lackautoimmune activity, or are minimally cross-reactive. Such molecularmimetics are useful in vaccine compositions and in eliciting antibodiesfor diagnostic or therapeutic applications, as described further below.Molecular mimetics include, but are not limited to, small organiccompounds; nucleic acids and nucleic acid derivatives; saccharides oroligosaccharides; peptide mimetics including peptides, proteins, andderivatives thereof, such as peptides containing non-peptide organicmoieties, synthetic peptides which may or may not contain amino acidsand/or peptide bonds, but retain the structural and functional featuresof a peptide ligand, and peptoids and oligopeptoids which are moleculescomprising N-substituted glycine, such as those described by Simon etal. (1992) Proc. Natl. Acad. Sci. USA 89:9367; and antibodies, includinganti-idiotype antibodies. Methods for the identification and productionof molecular mimetics are described more fully below.

The term “antibody” encompasses polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, F(ab′)₂ fragments, F(ab) molecules, Fv fragments,single domain antibodies, chimeric antibodies and functional fragmentsthereof which exhibit immunological binding properties of the parentantibody molecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited by the manner in which it is made. The term encompasses wholeimmunoglobulin molecules, as well as Fab molecules, F(ab′)₂ fragments,Fv fragments, and other molecules that exhibit immunological bindingproperties of the parent monoclonal antibody molecule. Methods of makingpolyclonal and monoclonal antibodies are known in the art and describedmore fully below.

An “antigen” is defined herein to include any substance that may bespecifically bound by an antibody molecule. An “immunogen” is an antigenthat is capable of initiating lymphocyte activation resulting in anantigen-specific immune response.

By “epitope” is meant a site on an antigen to which specific B cells andT cells respond. The term is also used interchangeably with “antigenicdeterminant” or “antigenic determinant site.” A peptide epitope cancomprise 3 or more amino acids in a spatial conformation unique to theepitope. Generally, an epitope consists of at least 5 such amino acidsand, more usually, consists of at least 8–10 such amino acids. Methodsof determining spatial conformation of amino acids are known in the artand include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance spectroscopy. Furthermore, the identificationof epitopes in a given protein is readily accomplished using techniqueswell known in the art. See, e.g., Geysen et al. (1984) Proc. Natl. Acad.Sci. USA 81:3998 (general method of rapidly synthesizing peptides todetermine the location of immunogenic epitopes in a given antigen); U.S.Pat. No. 4,708,871 (procedures for identifying and chemicallysynthesizing epitopes of antigens); and Geysen et al. (1986) MolecularImmunology 23:709 (technique for identifying peptides with high affinityfor a given antibody). Antibodies that recognize the same epitope can beidentified in a simple immunoassay showing the ability of one antibodyto block the binding of another antibody to a target antigen.

A “unique MenB epitope” is defined herein as an epitope present on aMenB bacterium, wherein antibodies directed toward the epitope arecapable of binding specifically to MenB and not cross reacting, orminimally cross reacting, with sialic acid residues present on thesurface of host tissue. Immunogens containing or mimicking one or more“unique MenB epitopes” are thus useful in vaccines for prevention ofMenB disease, and will not elicit an autoimmune response, or poseminimal risk of eliciting an autoimmune response.

An antibody displays “functional activity” against a MenB organism whenthe antibody molecule exhibits complement-mediated bactericidal activityand/or opsonic activity against MenB as determined using the assaysdescribed herein.

An antibody specific for a “unique” MenB epitope “lacks autoimmuneactivity,” and/or is “not autoreactive” when the subject antibody doesnot exhibit cross-reactive immunological binding properties withpolysialic acid in host tissue as determined using the binding assaysdescribed herein.

An antibody specific for a “unique” MenB epitope is “not autoreactive”when the subject antibody requires approximately ten times greaterantibody concentration to exhibit binding to polysialic acid in hosttissues, compared to a known cross-reactive auto antibody consideredpositive in the binding assays described herein. (For example, comparebinding of SEAM-12 to binding of SEAM-35 in FIG. 6). Thus, the termencompasses those antibodies that are not autoreactive or minimallyautoreactive in the binding assays described herein.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to non-covalent interactions of the type whichoccur between an immunoglobulin molecule and an antigen for which theimmunoglobulin is specific.

By “purified” and “isolated” is meant, when referring to a polypeptide,antibody or nucleotide sequence, that the indicated molecule is presentin the substantial absence of other biological macromolecules of thesame type. The terms “purified” and “isolated” as used herein preferablymean at least 75% by weight, more preferably at least 85% by weight,more preferably still at least 95% by weight, and most preferably atleast 98% by weight, of biological macromolecules of the same type arepresent. Similarly, an “isolated” antibody is an antibody separated froma mixed population of antibodies, such as from antisera raised against amolecule of interest.

“Homology” refers to the percent of identity between two polynucleotideor polypeptide moieties. The correspondence between two or moresequences can be determined by techniques known in the art. For example,homology can be determined by a direct comparison of the sequenceinformation between two polypeptide molecules. Two peptide sequences are“substantially homologous” when at least about 60% (preferably at leastabout 80%, and most preferably at least about 90%) of the amino acidsmatch.

II. Modes of Carrying Out the Invention

The present invention is based on the discovery of novel functionalantibodies directed against MenB. The antibodies do not cross-react, orare minimally cross-reactive with polysialic acid in host tissue asdetermined using the assays described herein, and hence the antibodieshave a lower risk of evoking autoimmune activity than antibodies thatare highly cross-reactive with host tissue. The antibodies can be usedto identify molecular mimetics of unique epitopes found on the surfaceof MenB. The antibodies and/or mimetics can be used in vaccinecompositions to treat and/or prevent MenB and E. coli K1 disease, aswell as in diagnostic compositions for the identification of MenB and E.coli K1 bacteria.

As explained above, the native capsular polysaccharide of MenB, termed“MenB PS” herein, is poorly immunogenic in humans and other mammaliansubjects. Furthermore, native MenB PS can elicit the production ofautoantibodies and, hence, may be inappropriate for use in vaccinecompositions. Thus, the present invention uses antibodies preparedagainst MenB PS derivatives. These antibodies are selected based ontheir ability to exhibit functional activity against MenB bacteria,wherein the functional activity is important in conferring protectionagainst MenB disease. The antibodies are also selected on the basis ofshowing minimal or undetectable autoimmune activity.

More particularly, MenB PS derivatives were prepared for use inobtaining the antibody molecules of the present invention. Thederivatives generally comprise C₃–C₈ acyl substitutions of sialic acidresidue N-acetyl groups of the native molecule. Particularly preferredMenB PS derivatives comprise the substitution of N-propionyl groups forN-acetyl groups of native MenB PS and are termed “NPr-MenB PS” herein.Such derivatives and methods for synthesizing the same are described ine.g., U.S. Pat. No. 4,727,136 and EP Publication No. 504,202 B, both toJennings et al.

The C₃–C₈ acyl derivatives can be made by first treating native MenB(obtained from e.g., N. meningitidis cultures) in the presence of astrong base to quantitatively remove the N-acetyl groups and to providea reactive amine group in the sialic acid residue parts of the molecule.The deacylated MenB PS fragments are then N-acylated. For example, inthe case of NPr-MenB PS, the deacylated molecule is N-propionylatedusing a source of propionyl groups such as propionic anhydride orpropionyl chloride, as described in U.S. Pat. No. 4,727,136 to Jenningset al. The extent of N-acylation can be determined using, for example,NMR spectroscopy. In general, reaction conditions are selected such thatthe extent of N-acylation is at least about 80%.

In order to increase the immunogenicity of the MenB PS derivatives, thederivatives can be conjugated to a suitable carrier molecule to provideglycoconjugates. Particularly, N-acylated MenB PS glycoconjugatepreparations having well defined and controlled structuralconfigurations can be formed from intermediate sized N-acylated MenBoligosaccharides as described below.

Thus, a group of N-acylated MenB PS glycoconjugates, an example of whichis termed “COMJ-2” herein can be prepared as follows. An N-acylated MenBPS preparation, having substantially 100% N-acylated MenB sialic acidresidues, as determined by e.g., NMR analysis, can be fragmented undermild acidic conditions to provide a population of oligosaccharidemolecules of varying sizes. The fragmented products are sizefractionated using for example standard ion exchange chromatographictechniques combined with e.g., stepwise salt gradients, to providefractions of N-acylated MenB molecules of homogenous sizes. Fractionscontaining intermediate sized oligosaccharides e.g., with an average Dpor about 5 to about 22, preferably 10 to about 20, and more particularlyabout 12 to about 18, are chemically end-activated at the non reducingtermini and conjugated to protein carriers by a reductive aminationtechnique to provide the CONJ-2 glyoconjugation. Successful conjugationcan be determined by, e.g., gel filtration, and the final saccharide toprotein ratio (w/w) assessed by colorimetric assay.

Glycoconjugates formed from MenB PS derivatives, such as the CONJ-2 arethen used herein to elicit the formation of anti-saccharide antibodiesin an immunized host a subset of such antibodies should bind to MenBbacteria should not cross-react, or be minimally cross-reactive withhost tissue sialic acid residues as determined the binding assaysdescribed herein. The antibodies can be fully characterized with respectto isotype the antigenic specificity, functional activity and crossreactivity with host tissue.

For example, mammalian subjects, conveniently standard laboratoryanimals such as rodents and rabbits, can be summarized with compositionscontaining the glycoconjugates along with a suitable adjuvant to elicitthe production of polyclonal sera. Groups of animals are generallyimmunized and boosted several times with the compositions. Antisera fromimmunized animals can be obtained, and polyclonal sera that does notcross-react with host tissue can be obtained using in-situ absorption orconventional affinity chromatography techniques. Successfulglycoconjugate antigens can be identified by their ability to elicit asubstantial IgG anti MenB PS derivative antibody response,characteristic of a T-cell dependent antigen. Conjugates that are foundto be highly immunogenic and produce predominantly IgG antibodies areparticularly preferred for use in the methods of the present invention.

MenB PS derivatives that are capable of eliciting the formation ofbactericidal antisera are suitable for use in the production ofmonoclonal antibodies. More particularly, the process used to providethe various MenB PS derivative conjugates is designed to producesuperior immunogens presenting unique saccharide-associated epitopesthat mimic those found on the surface of MenB organisms and areexpressed minimally in the host. The MenB PS derivatives describedherein and thus capable of eliciting the production of MenB-specificantibodies which can be use directly in the selective or therapeuticpharmaceutical preparation or, preferably, used to search for mimeticsof MenB polysaccharide antigens that will provide unique epitopes foranti-MenB vaccines.

Thus is one embodiment of the invention, selected MenB derivatives areused to provide monoclonal antibodies and functional equivalentsthereof. The term “functional equivalent” with respect to a particularthat: (a) cross-blocks an exemplified monoclonal antibodies kindsselectively to the MenB PS derivative or glycoconjugate in question; (c)does not cross-react, or minimally cross-reacts, with host PSA asdetermined using the binding assays described herein; and, optionally,activity (e.g., complement-mediated bactericidal and/or opsonicactivity) against MenB bacterial cells as determined by standard assaysdescribed below. Further, as used herein with regard to a particularmonoclonal antibody producing hybridoma of the invention, the term“progeny” is intended to include all derivatives, issue, and offspringof the parent hybridoma that produce the monoclonal antibody produced bythe parent, regardless of generation or karyotypic identity.

Monoclonal antibodies are prepared using standard techniques, well knownin the art, such as by the method of Kohler and Milstein, Nature (1975)256:495, or a modification thereof, such as described by Buck et al.(1982) In Vitro 18:377. Typically, a mouse or rat is immunized with theMenB PS derivative conjugated to a protein carrier, boosted and thespleen (and optionally several large lymph nodes) removed anddissociated into single cells. If desired, the spleen cells may bescreened (after removal of non-specifically adherent cells) by applyinga cell suspension to a plate or well coated with the antigen. B-cells,expressing membrane-bound immunoglobulin specific for the antigen, willbind to the plate, and will not be rinsed away with the rest of thesuspension. Resulting B-cells, or all dissociated spleen cells, are theninduced to fuse with myeloma cells to form hybridomas. Representativemurine myeloma lines for use in the hybridizations include thoseavailable from the American Type Culture Collection (ATCC).

More particularly, somatic cell hybrids can be prepared by the method ofBuck et al., (supra), using the azaguanine resistant, non-secretingmurine myeloma cell line P3X63-Ag8.653 (obtainable from the ATCC). Thehybridoma cell lines are generally cloned by limiting dilution, andassayed for the production of antibodies which bind specifically to theimmunizing antigen and which do not bind to unrelated antigens. Theselected monoclonal antibody-secreting hybridomas are then culturedeither in vitro (e.g., in tissue culture bottles or hollow fiberreactors), or in vivo (e.g., as ascites in mice).

Hybridoma supernatant can be assayed for anti-MenB PS derivativereactive antibody using, for example, either solid phase ELISA or anindirect immunofluorescence assay with the immunizing MenB PS derivativeor with native MenB PS (NAc-MenB PS). The selectivity of monoclonalantibodies secreted by the hybridomas can be assessed using competitivespecific binding assays, such as inhibition ELISA, or the like. Forexample, antibody molecules, either diluted in buffer, or buffercontaining soluble MenB PS derivatives or NAc-MenB PS, are reacted in anELISA vessel in the presence of bound MenB PS derivatives. Afterwashing, bound antibody is detected by labeled anti-Ig (anti-IgM, IgGand IgA) as the secondary antibody. Antibodies that are inhibited by thesoluble MenB PS derivatives can be considered specific and, thus areselected for further study including, isotyping and additional screeningfor cross-reactivity, functional activity, and autoreactivity.

Specifically, partially purified monoclonal antibody molecules can beindividually evaluated for their ability to bind to host cells whichexpress polysialic acid residues on their cell surfaces. Such cellsrepresent surrogate targets for the detection of antibodies that exhibitautoimmune activity. One target comprises the human neuroblastoma cellline, CHP-134, which expresses long chain α2–8 polysialic acid (NCAM) onits cell surface, as described by Livingston et al. (1988) J. Biol.Chem. 263:9443. Other suitable targets include, but are not limited to,newborn brain cells, tissues derived from e.g., kidney, heart and theolfactory nerve, cultured saphenous vein endothelial cells, cytotoxic Tlymphocytes and natural killer (NK) cells. See, e.g., Brandon et al.(1993) Intl. J. Immunopathology and Pharmacology 6:77. Monoclonalantibody molecules obtained from the hybridomas can be added to suitabletest cell populations in culture, and the potential binding of themonoclonals to the cellular targets detected and quantified directlyusing labeled monoclonals, or indirectly using an appropriately labeledsecondary reagent that reacts specifically with each monoclonal antibody(e.g., Staphylococcal Protein A and G and anti-murine antibodymolecules). Antibodies that do not cross-react with test host tissue PSAor that display minimal reactivity are not considered autoreactive forpurposes of the present invention. Thus, these antibodies areappropriate for further use. In addition, some antibodies that showbinding with test tissue, which binding is not affected by pre-treatmentof the test cells with neuraminidase, may also be appropriate forfurther use. Autoreactivity of such antibodies is termed “indeterminate”herein.

Functional activity can be determined by assessing complement-mediatedbactericidal activity and/or opsonic activity. In particular,complement-mediated bactericidal activity of the antibodies can beevaluated using standard assays such as those described by Gold et al.(1970) Infect. Immun. 1:479, Westerink et al. (1988) Infect. Immun.56:1120, Mandrell et al. (1995) J. Infect. Dis. 172:1279, and Granoff etal. (1995) Clin. Diagn. Laboratory Immunol, 2:57. In these assays, N.meningitidis is reacted with a complement source as well as with theantibody to be tested. Bacterial counts are done at various samplingtimes. Those antibodies that demonstrate complement-mediatedbactericidal activity, as demonstrated by a minimum of a 50% reductionin viable bacterial cell counts determined after sixty minutesincubation with antibody and complement, as compared to colony counts attime zero, are considered to exhibit bactericidal activity for purposesof the present invention and are suitable for further use.

Complement-mediated bacteriolysis is thought to be the major mechanismresponsible for host protection against invasive Meningococcal disease.However, evidence also supports an important protective role foropsonization (see, e.g., Bjerknes et al. (1995) Infect. Immun. 63:160).Accordingly, the opsonic activity of the antibodies produced herein canbe evaluated as a second measure, or as an alternative measure, toassess functional activity. Results from opsonic assays can be used tosupplement bactericidal data, and to help in the selection of antibodiescapable of conferring protection. Evaluation of optimal activity is alsoparticularly useful herein for the evaluation of the murine monoclonalantibodies of the invention which have an IgGl isotype. Murine IgGl (incontrast to human IgGl) is ineffective in activation of complement.Thus, murine IgGl antibodies do not activate complement-mediatedbacteriolysis of MenB in the above described assays. However, functionalactivity of IgGl anti-NPr-MenB PS monoclonal antibodies can be accessedby opsonization in the absence of complement.

A variety of opsonic assay methods are known in the art and can be usedto evaluate functional activity of the monoclonal antibodies of thepresent invention. Such standard assays include those described bySjursen et al. (1987) Acta Path. Microbiol. Immunol. Scand., Sec. C95:283, Halstensen et al. (1989) Scand. J. Infect. Dis. 21:267, Lehmannet al. (1991) APMIS 99:769, Halstensen et al. (1991) NIPH Annals 14:157,Fredlund et al. (1992) APMIS 100:449, Guttormsen et al. (1992) Infect.Immun. 60:2777, Guttormsen et al. (1993) J. Infec. Dis. 167:1314,Bjerknes et al. (1995) Infect. Immun. 63:160, Hayrinen et al. (1995) J.Infect. Dis. 171:1481, de Velasco et al. (1995) J. Infect. Dis. 172:262,and Verheul, A. F. M. (1991) “Meningococcal LPS DerivedOligosaccharide-Protein Conjugate Vaccines, Immunochemical andImmunological Aspects,” Thesis, Utrecht University, The Netherlands, pp.112–135.

Selected monoclonal antibodies of interest can be expanded in vitro,using routine tissue culture methods, or in vivo, using mammaliansubjects. For example, pristane-primed mice can be inoculated with logphase hybridoma cells in PBS for ascites production. Ascites fluid canbe stored at −70° C. prior to further purification.

It may be desirable to provide chimeric antibodies, especially if theantibodies are to be used in preventive or therapeutic pharmaceuticalpreparations, such as for providing passive protection against MenB., aswell as in MenB diagnostic preparations. Chimeric antibodies composed ofhuman and non-human amino acid sequences may be formed from the mousemonoclonal antibody molecules to reduce their immunogenicity in humans(Winter et al. (1991) Nature 349:293; Lobuglio et al. (1989) Proc. Nat.Acad. Sci. USA 86:4220; Shaw et al. (1987) J Immunol. 138:4534; andBrown et al. (1987) Cancer Res. 47:3577; Riechmann et al. (1988) Nature332:323; Verhoeyen et al. (1988) Science 239:1534; and Jones et al.(1986) Nature 321:522; EP Publication No. 519,596, published 23 Dec.1992, and U.K. Patent Publication No. GB 2,276,169, published 23 Sep.1994).

Antibody molecule fragments, e.g., F(ab′)₂, Fv, and sFv molecules, thatare capable of exhibiting immunological binding properties of the parentmonoclonal antibody molecule can be produced using known techniques.Inbar at al. (1973) Proc. Nat. Acad. Sci. USA 69:2659; Hochman et al.(1975) Biochem 15:2706; Ehrlich et al. (1980) Biochem 19:4091; Huston etal. (1986) Proc. Nat. Acad. Sci. USA 85:(16):5879; and U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

In the alternative, a phage-display system can be used to expand themonoclonal antibody molecule populations in vitro. Salkl, et al. (1986)Nature 324:163; Scharf et al. (1985) Science 133:1076; U.S. Pat. Nos.4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392; Barbus,III et al. (1995) Methods: Comp. Meth Enzymol 8:94; Barbas, III et al.(1991) Proc Natl Acad Sci USA 88:7978.

Once generated, the phage-display library can be used to improve theimmunological binding affinity of the Fab molecules using knowntechniques. See, e.g., Fagina et al. (1994) J. Mol. Biol. 239:68.

The ongoing sequences for the heavy and light chain portions of the Fabmolecules selected from the image display library can be isolated orsynthesized, and cloned into any suitable vector or replicon forexpression. Any suitable expression system can be used, including forexample, bacterial, yeast, insect, amphibian and mammalian systems.Expression systems in bacteria include those described in Chang et al.(1978) Nature 275:615, Goeddel et al. (1979) Nature 281:544, Goeddel etal. (1980) Nucleic Acids Res. 8:4057, European Application No. EP36,776, U.S. Pat. No. 4,551,433, deBoer et al. (1983) Proc. Natl. Acad.Sci. USA 80:21–25, and Siebenlist et al. (1980) Cell 20:269.

Expression systems in yeast include those described in Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA 75:1929, Ito et al. (1983) J.Bacteriol. 153:163, Kurtz et al. (1986) Mol. Cell. Biol. 6:142, Kunze etal. (1985) J. Basic Microbiol. 25:141, Gleeson et al. (1986) J. Gen.Microbiol. 132:3459, Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302,Das et al. (1984) J. Bacteriol. 158:1165, De Louvencourt et al. (1983)J. Bacteriol. 154:737, Van den Berg et al. (1990) Bio/Technology 8:135,Kunze et al. (1985) J. Basic Microbiol. 25:141, Cregg et al. (1985) Mol.Cell. Biol. 5:3376, U.S. Pat. Nos. 4,837,148 and 4,929,555, Beach et al.(1981) Nature 300:706, Davidow et al. (1985) Curr. Genet. 10:380,Gaillardin et al. (1985) Curr. Genet. 10:49, Ballance et al. (1983)Biochem. Biophys. Res. Commun. 112:284–289, Tilburn et al. (1983) Gene26:205–221, Yelton et al. (1984) Proc. Natl. Acad. Sci. USA81:1470–1474, Kelly et al. (1985) EMBO J. 4:475479; European ApplicationNo. EP 244,234, and International Publication No. WO 91/00357.

Expression of heterologous genes in insects can be accomplished asdescribed in U.S. Pat. No. 4,745,051, European Application Nos. EP127,839 and EP 155,476, Vlak et al. (1988) J. Gen. Virol. 69:765–776,Miller et al. (1988) Ann. Rev. Microbiol. 42:177, Carbonell et al.(1988) Gene 73:409, Maeda et al. (1985) Nature 315:592–594,Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8:3129, Smith et al.(1985) Proc. Natl. Acad. Sci. USA 82:8404, Miyajima et al. (1987) Gene58:273, and Martin et al. (1988) DNA 7:99. Numerous baculoviral strainsand variants and corresponding permissive insect host cells from hostsare described in Luckow et al. (1988) Bio/Technology 6:47–55, Miller etal. (1986) GENERIC ENGINEERING, Setlow, J. K. et al. eds., Vol. 8,Plenum Publishing, pp. 277–279, and Maeda et al. (1985) Nature315:592–594.

Mammalian expression can be accomplished as described in Dijkema et al.(1985) EMBO J. 4:761, Gorman et al. (1982) Proc. Natl. Acad. Sci. USA79:6777, Boshart et al. (1985) Cell 41:521, and U.S. Pat. No. 4,399,216.Other features of mammalian expression can be facilitated as describedin Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980) Anal.Biochem. 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,4,560,655 and Reissued U.S. Pat. No. RE 30,985, and in InternationalPublication Nos. WO 90/103430, WO 87/00195.

Any of the above-described antibody molecules can be used herein toprovide anti-MenB therapeutic or preventive pharmaceutical agents.Additionally, “humanized” antibody molecules, comprising antigen-bindingsites derived from the instant murine monoclonal antibodies, can beproduced using the techniques described above.

The anti-MenB antibodies of the present invention, described above, areconveniently used as receptors to screen diverse molecular libraries inorder to identify molecular mimetics of unique epitopes from MenB.Methods for identifying mimetics in molecular libraries generallyinvolve the use of one or more of the following procedures: (1) affinitypurification with an immobilized target receptor; (2) binding of asoluble receptor to tethered ligands; and (3) testing soluble compoundsdirectly in antigen competition assays or for biological activity.Molecules screened for molecular mimics include but are not limited tosmall organic compounds, combinatorial libraries of organic compounds,nucleic acids, nucleic acid derivatives, saccharides oroligosaccharides, peptoids, soluble peptides, peptides tethered on asolid phase, peptides displayed on bacterial phage surface proteins,bacterial surface proteins or antibodies, and/or peptides containingnon-peptide organic moieties.

For example, libraries of diverse molecular species can be made usingcombinatorial organic synthesis. See, e.g., Gordon et al. (1994) J. Med.Chem. 37:1335. Examples include but are not limited to oligocarbamates(Cho et al. (1993) Science 261:1303); peptoids such as N-substitutedglycine polymers (Simon et al. (1992) Proc. Natl. Acad. Sci. USA89:9367); and vinylogous polypeptides (Hagihara et al. (1992) J. Am.Chem. Soc. 114:6568).

A variety of approaches, known in the art, can be used to track thebuilding blocks as they are added during synthesis so that the historyof individual library members can be determined. These approachesinclude addressable location on a photolithographic chip(oligocarbamates), a deconvolution strategy in which “hits” areidentified through recursive additions of monomers to partiallysynthesized libraries (peptoids, peptides), and coding combinatoriallibraries by the separate synthesis of nucleotides (Nielsen et al.(1993) J. Am. Chem. Soc. 115: 9812) or other organic moieties (Ohlmeyeret al. (1993) Proc. Natl. Acad. Sci. USA 90:10922) (“tags”). The codedtags associated with each library member can then be decoded after amimetic has been selected. For example, nucleic acid tags can be decodedby DNA sequencing.

Peptoid combinatorial libraries are particularly useful for identifyingmolecular mimetics of unique MenB epitopes. Peptoids are oligomers ofN-substituted glycine (Simon et al. (1992) Proc. Natl. Acad. Sci. USA89:9367) and can be used to generate chemically diverse libraries ofnovel molecules. The monomers may incorporate t-butyl-based side-chainand 9-fluorenylmethoxy-carbonyl α-amine protection. The assembly ofmonomers into peptoid oligomers can be performed, for example, on asolid phase using the “submonomer method” of Zuckermann et al. (1991) J.Am. Chem. Soc. 114:10646. In this method, syntheses are conducted withRink amide polystyrene resin (Rink et al. (1987) Tetrahedron Lett.28:3787). Resin-bound amines are bromoacetylated by in situ activationof bromoacetic acid with diisopropylcarbodiimide. Subsequently, theresin-bound bromoacetamides are displaced by addition of an amine. Theamines may incorporate t-butyl-based protection of additional reactivegroups. This two-step cycle is repeated until the desired number ofmonomers is added. The oligopeptide is then released from the resin bytreatment with 95% trifluroacetic acid/5% water. The syntheses areperformed, preferably, using a robotic synthesizer. See, e.g.,Zuckermann et al. (1992) Pept. Protein Res. 40:498. In the alternative,oligomerization of the peptoid monomers may be performed by in situactivation by either benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorphosphate or bromotris(pyrrolidino)phosphoniumhexafluorophosphate. In this alternative method, the other steps areidentical to conventional peptide synthesis usingα-(9-fluorenylmethoxycarbonyl) amino acids (see, e.g., Simon et al.(1992), supra).

Once the peptoid libraries are generated, they can be screened by, e.g.,adding the monoclonal antibodies of the present invention, along withvarious pools of the combinatorial peptoids, to wells of microtiterplates coated with MenB PS derivatives or MenB bacteria, either alone oras glycoconjugates. After a period of incubation and a wash to removeunbound antibody, the presence of bound antibody is determined bystandard ELISA assays. See, e.g., Harlow & Lane, Antibodies: ALaboratory Manual (1988), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 553. Wells that do not contain bound antibody indicate thepresence of peptoid mimetics that bind to the antibody. The particularidentities of the peptoid mimetics in the pools are determined byrecursively adding back monomer units to partially synthesized membersof the libraries. Zuckermann et al. (1994) J. Med. Chem. 37:2678.

Peptide libraries can also be used to screen for molecular mimetics ofunique epitopes of MenB using the anti-MenB antibodies of the presentinvention. Such libraries are based on peptides such as, but not limitedto, synthetic peptides that are soluble (Houghten (1985) Proc. Natl.Acad. Sci. USA 82:5131) or tethered to a solid support (Geysen et al.(1987) Immunol. Methods 102:259; U.S. Pat. No. 4,708,871) and peptidesexpressed biologically as fusion proteins (Scott et al. (1990) Science249:386). For a review of peptide combinatorial libraries, see, e.g.,Gallop et al. (1994) J. Med. Chem. 37:1233.

For example, random soluble peptides, having known sequences, can besynthesized on solid supports and members of the library separated fromeach other during the repetitive coupling/deprotection cycles inindividual labeled polypropylene bags (Houghten (1985) Proc. Natl. Acad.Sci. USA 82:5131). Following synthesis, the peptides are cleaved fromthe solid support and identified by the label on the polypropylene bag.The synthetic peptide library generated using this method can bescreened for binding to an antibody having the desired properties byadsorbing individual peptides to microtiter plate wells and determiningantibody binding using standard ELISA assays.

Large, libraries of potential peptide mimetics can also be constructedby concurrent synthesis of overlapping peptides as described in U.S.Pat. No. 4,708,871. to Geyser. The synthetic peptides can be tested forinteraction with the antibodies by ELISA while still attached to thesupport used for synthesis. The solid support is generally apolyethylene or polypropylene rod onto which is graft polymerized avinyl monomer containing at least one functional group to producepolymeric chains on the carrier. The functional groups which aresequentially reacted with amino acid residues in the appropriate orderto build the desired synthetic peptide using conventional methods ofsolid phase peptide chemistry. For example, peptide sequences can bemade by parallel synthesis on polyacrylic acid-grafted polyethylene pinsarrayed in microtiter plates, as described in Geyser et al. (1987) J.Immunol. Methods 102:259. Such libraries can be screened by, e.g.,adding antibody to wells containing the peptide-pins. After washingunbound antibody from the cells, the presence of bound antibody can bedetected using an ELISA assay.

Peptide mimetics that interact with the antibodies of the presentinvention can also be identified using biological expression systems.See, e.g., Christian et al. (1992) J. Mol. Biol. 227:711; Devlin et. al.(1990) Science 249:404; Cwirla et. al. (1990) Proc. Acad. Sci. USA87:6378; Gallop et al. (1994) J. Med. Chem 37:1233. Using such systems,large libraries of peptide sequences can be screened for molecules thatbind the antibodies of the present invention. This approach also allowsfor simple molecular characterization of identified mimetics since DNAencoding the peptides can be readily sequenced. Additionally, raremimetics can be amplified through several rounds ofselection/amplification.

For example, phage-display libraries can be produced by insertingsynthetic DNA pieces, encoding random peptide sequences, near the 5′-endof the gene encoding the pIII or pVIII protein of the filamentousbacterial phage m13, fd, or f1 (Parmley et al. (1988) Gene 73:305; Smithet al. (1993) Meth. Enzymol. 217:228). The phage, phagemid, or plasmidDNA containing the gene and randomized extension is then used totransform a suitable host such as E. coli or E. coli coinfected with ahelper phage. The phage isolated from the culture carry pIII (1–5copies) or pVIII (˜4000 copies) surface proteins having the randomizedpeptide sequences extending from the amino terminus. Phage can bepurified by, e.g., affinity purification by biotinylating the receptorantibodies of the present invention, incubating the phage with thebiotinylated receptor and reacting the phage on streptavidin-coatedplates. Bound phage are eluted and amplified by infecting a suitablehost on agar medium and subjected to further rounds of affinitypurification. Phage from later rounds of affinity purification can becloned and propagated, their DNAs sequenced to determine the amino acidsequences of their expressed peptide and their binding to MenBantibodies assessed by ELISA or by a variety of other screeningprocedures, well known in the art.

Combinatorial libraries of human Fab antibodies can also be displayed onphage surface proteins to select useful molecular mimetics for useherein. Preparation of such libraries has been described hereinabove.See, e.g., Burton et al. (1994) Adv. Immunol. 57:191 for a review ofsuch techniques.

Molecular mimetics of MenB unique epitopes can also be identified usingthe anti-MenB antibodies of the present invention in those methodsdescribed by Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865. TheCull technique utilizes the DNA binding protein, LacI, to form a linkbetween peptide and its encoding DNA sequence. In this method, DNAencoding randomized peptides is appended to the 3′-end of the LacI genepresent on a plasmid. The plasmid also contains the DNA binding site forLacI, lacO. When Lacd is expressed from the plasmid in a suitable host(e.g. E. coli), it binds tightly to lacO. Thus, when the cells arelysed, each copy of LacI that displays a randomized peptide at itscarboxyl terminus is associated with the DNA encoding it. Methods forscreening, amplifying, and sequencing these “peptides-on-plasmids”libraries are the same as those used in phage display, as describedabove.

Molecular mimetics can also be identified using the anti-MenB antibodiesin in vitro, cell-free systems such as the system described byMattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022. In thisapproach, nascent peptides are displayed in polysome complexes andconstruction of libraries, expression of the peptides, and screening iscarried out in a cell-free system. Peptides displayed on polysomes canbe screened using, for example, an affinity purification/amplificationscreening procedure where the MenB-specific antibody/receptor isimmobilized, e.g., on a plastic plate.

Molecules used in the libraries above can be manipulated in order toform more stable conformations and thus enhance identification of usefulmolecular mimetics. For example, cysteine residues can be incorporatedin the randomized sequences to form disulfide loops (O'Neal et al.(1992) Proteins 14:509) and protein scaffolds can be used to displayrandomized peptides in internal loop segments (Freimuth et al. (1990) J.Biol. Chem. 265:896; Sollazzo et al. (1990) Prot. Engin. 4:215).

Anti-idiotypic antibodies can also be produced using the anti-MenBantibodies of the present invention for use as molecular mimetics ofunique epitopes of MenB. For a review of anti-idiotype antibodies, see,e.g., Kieber-Emmons et al. (1986) Int. Rev. Immunol. 1:1. In thisregard, the pocket or cleft formed by the heavy and light chains of anantibody is often intimately involved in antigen binding. This region,called the paratope, is an “internal image” of the antigen surface boundby the antibody. An antibody directed against the paratope is one ofseveral potential anti-idiotypic antibodies and can be a mimetic of theantigen. Randomized peptide loops of the heavy and light chains occurnaturally as part of the generation of antibody diversity.

Anti-MenB monoclonal antibodies of the present invention can be used toelicit anti-idiotype antibody production and to select anti-idiotypesbearing the “image” of the antigen, using the techniques described ine.g., Westerink et al. (1988) Infect. Immun. 56:1120.

In one embodiment, a combinatorial library of phage-display antibodies,as described above, are screened using the anti-MenB monoclonalantibodies of the present invention to identify mimetic antibodies, i.e.phage-display Fab anti-idiotypic antibodies.

Anti-idiotype antibodies produced can be easily tested for their abilityto elicit anti-MenB antibody production in standard laboratory animalmodels. The variable genes of the anti-idiotype antibodies can besequenced to identify peptide vaccine candidates.

Additionally, combinatorial libraries of oligonucleotides (DNA, RNA, andmodified nucleotides) can be screened to find molecular mimetics thatbind to the non-autoreactive, anti-MenB antibodies of the presentinvention. Techniques for the production and use of such libraries arereviewed in e.g., Gold et al. (1995) Annu. Rev. Biochem. 64:763. Asystem, known as SELEX for Systematic Evolution of Ligands byExponential enrichment, can be used for rapidly screening vast numbersof oligonucleotides for specific sequences that have desired bindingaffinities and specificities toward the anti-MenB antibodies. (Tuerk etal. (1990) Science 249:505). For example, immobilized non-autoreactiveMenB monoclonal antibodies can be used to affinity purify specificbinding oligonucleotides from a combinatorial library. The boundoligonucleotides are released from the immobilized antibodies by addinga competitive ligand or lowering the pH. The released oligonucleotidesare either amplified directly using the polymerase chain reaction orconverted to double stranded DNA using reverse transcriptase (Tuerk etal., 1990, supra). This is followed by additional rounds of selectionand amplification until the desired mimetic is obtained. The sequencesof the oligonucleotide mimetics are determined by DNA sequencing.

Once putative molecular mimetics are identified, they are tested fortheir ability to elicit functionally active (e.g., bactericidal and/oropsonic) antibodies which lack autoreactivity or have minimalautoreactivity, as described above. Molecular mimetics that have theseproperties are appropriate for further use, for example, in vaccinecompositions.

The anti-MenB monoclonal antibodies can also be used to investigate thebactericidal and/or opsonic function of antibodies of differentspecificities, as well as to identify the molecular nature of the uniqueepitopes on the MenB bacterial surface that are not cross-reactive withhost PSA. Furthermore, the anti-MenB antibodies can be used to isolatefractions of MenB bacteria or MenB PS derivatives. Once isolated, thecritical epitopes reactive with the anti-MenB antibodies can becharacterized and employed directly in oligosaccharide protein conjugatevaccines or to model synthetic saccharides or mimetics for use invaccines.

Molecular mimetics identified using the functionally active anti-MenBantibodies of the invention can be used to generate antibody reagentsfor use in diagnostic assays. For example, antibodies reactive with themolecular mimetics can be used to detect bacterial antigen in biologicalsamples using immunodiagnostic techniques such as competition, directreaction, or sandwich type assays. Such assays include Western blots;agglutination tests; enzyme-labeled and mediated immunoassays, such asELISAs; biotin/avidin type assays; radioimmunoassays;immunoelectrophoresis; immunoprecipitation, and the like.

In addition, molecular mimetics, unique (e.g., non-autoimmune) Men Bepitopes identified using the molecular mimetics and anti-id monoclonalantibodies can be used herein in vaccine compositions for the preventionof MenB disease in vaccinated subjects.

The vaccine compositions can comprise one or more of the anti-idmonoclonal antibodies, molecular mimetics or non-autoimmune epitopes ofMenB. The vaccines may also be administered in conjunction with otherantigens and immunoregulatory agents, for example, immunoglobulins,cytokines, lymphokines, and chemokines, including but not limited toIL-2, modified IL-2 (cys125→ser125), GM-CSF, IL-12, γ-interferon, IP-10,MIP1β and RANTES.

The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents of buffering substances, and the like, may be presentis any vehicles.

Adjuvants may also be used to enhance the effectiveness of the vaccines;Adjuvants can be added directly to the vaccine compositions or can beadministered separately, either concurrently with or shortly after,vaccine administration. Such adjuvants include, but are not limited to:(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% TWEEN 80(polyoxyethylenesorbitan monooleate), and 0.5% SPAN 85 (sorbitantrioleate) (optionally containing various amounts of MTP-PE (see below),although not required) formulated into submicron particles using amicrofluidizer such as Model 11Y microfluidizer (Microfluidics, Newton,Mass.), (b) SAF, containing 10% Squalane, 0.4% TWEEN 80(polyoxyethylenesorbitan monooleate), 5% pluronic-blocked polymer L121,and thr-MDP (see below) either microfluidized into a submicron emulsionor vortexed to generate a larger particle size emulsion, and (c) Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% TWEEN 80 (polyoxyethylenesorbitan monooleate), and one ormore bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, suchas Stimulon™(Cambridge Bioscience, Worcester, Mass.) may be used orparticle generated therefrom such as ISCOMs (immunostimulatingcomplexes); (4) Freund's Complete Adjuvant (FCA) and Freund's IncompleteAdjuvant (FICA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; and (6) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

In order to enhance the effectiveness of vaccine compositions formedfrom a molecular mimetic, it may be necessary to conjugate the mimeticto a carrier molecule. Such carrier molecules will not themselves inducethe production of harmful antibodies. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), inactive virus particles, CRM₁₉₇ (a nontoxic mutantdiphtheria toxin), and the like. Such carriers are well known to thoseof ordinary skill in the art. The mimetic conjugates are selected fortheir ability to express epitopes that closely resemble those found onthe surface of MenB bacterial cells. Suitable conjugates thus elicit theformation of antibodies that have functional activity against bacteria,and do not cross-react, or are minimally cross-reactive with polysialicacid in host tissue as determined using the binding assays describedherein.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes, or adsorbed to particles for enhanced adjuvant effect, asdiscussed above.

The vaccines will comprise an effective amount of the anti-id monoclonalantibody; molecular mimetic, peptide molecular mimetic or complexes ofproteins; or nucleotide sequences encoding the same, and any other ofthe above-mentioned components, as needed. By “an effective amount” ismeant an amount of a molecule which will induce an immunologicalresponse in the individual to which it is administered and poses aminimal risk of stimulating an autoimmune response in the individual.Such a response will generally result in the development in the subjectof a secretory, cellular and/or antibody-mediated immune response to thevaccine. Usually, such a response includes but is not limited to one ormore of the following effects; the production of antibodies from any ofthe immunological classes, such as immunoglobulins A, D, E, G or M; theproliferation of B and T lymphocytes; the provision of activation,growth and differentiation signals to immunological cells; expansion ofhelper T cell, suppressor T cell, and/or cytotoxic T cell and/or γδ Tcell populations.

Once formulated, the vaccines are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule.

Polynucleotides encoding DNA or RNA mimetics of the MenB PS can also beused in vaccines for nucleic acid immunization. In the alternative,polynucleotides encoding peptide mimetics can be used in nucleic acidimmunization. Such methods generally comprise the introduction of apolynucleotide encoding one or more of the desired molecules into a hostcell, for the in vivo expression of the nucleic acid molecules orproteins. The polynucleotide can be introduced directly into therecipient subject, such as by injection, inhalation or the like, or canbe introduced ex vivo, into cells which have been removed from the host.In the latter case, the transformed cells are reintroduced into thesubject where an immune response can be mounted against the moleculeencoded by the polynucleotide. Methods of nucleic acid immunization areknown in the art and disclosed in e.g., International Publication No. WO93/14778 (published 5 Aug. 1993); International Publication No. WO90/11092 (published 4 Oct. 1990); Wang et al. Proc. Natl. Acad. Sci. USA(1993) 90:4156; Tang et al. Nature (1992) 356:152; and Ulmer et al.Science (1993) 259:1745. Generally, the polynucleotide is administeredas a vector which has been encapsulated in a liposome and formulatedinto a vaccine composition as described above.

The anti-MenB monoclonal antibodies of the present invention, andfunctional equivalents thereof, can be used in pharmaceuticalcompositions to treat and/or prevent MenB and E. coli K1 disease inmammals. Such disease includes bacterial meningitis and sepsis, ininfants, children and adults. In this regard, the administration of ahighly-active, anti-MenB monoclonal antibody preparation to anindividual who is at risk of infection, or who has been recently exposedto the agent will provide immediate passive immunity to the individual.Such passive immunizations would be expected to be successful in bothnormal and immunocompromised subjects. Further, administration of suchmonoclonal antibody compositions can be used to provide antibody titerto MenB in a mammalian subject, either alone, or in combination withknown anti-MenB therapeutics.

The pharmaceutical compositions of the present invention generallycomprise mixtures of one or more of the above described anti-MenBmonoclonal antibodies, including Fab molecules, Fv fragments, sFvmolecules and combinations thereof. The compositions can be used toprevent MenB disease or to treat individuals following MenB infection.

Therapeutic uses of the pharmaceutical compositions involve bothreduction and/or elimination of the MenB infection agent from infectedindividuals, as well as the reduction and/or elimination of thecirculating MenB agent and the possible spread of the disease.

As described above in regard to the vaccine compositions of the presentinvention, the pharmaceutical compositions can be administered inconjunction with ancillary immunoregulatory agents such as IL-2,modified IL-2 (cyc125→ser125), GM-CSF, IL-12, γ-interferon, IP-10, MIP1βand RANTES.

The preparation of pharmaceutical composition containing or moreantibodies, antibody fragments, sFv molecules or combinations thereof,as the active ingredient is generally known to those of skill in theart. Once formulated, the compositions are conventionally administeredparenterally, e.g., by injection (either subcutaneously, intravenouslyor intramuscularly). Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications.

The pharmaceutical compositions are administered to the subject on betreated in a manner compatible with the dosage formulation and in anamount that will be prophylactically and/or therapeutically effective.The amount of the composition to be delivered, generally in the range offrom about 50 to about 10,000 micrograms of active agent per dose,depends on the subject to be treated, the capacity of the subject'simmune system to mount its own immune-responses, and the degree ofprotection desired. The exact amount necessary will vary depending onthe age and general condition of the individual to be treated, theseverity of the condition being treated and the mode of administration,among other factors. An appropriate effective amount can be readilydetermined by one of skill in the art. Thus, “an effective amount” ofthe pharmaceutical composition will be sufficient to bring abouttreatment or prevention of MenB disease symptoms, and will fall in arelatively broad range that can be determined through routine trials.

In addition, the pharmaceutical compositions can be given in a singledose schedule, or preferably in a multiple dose schedule. A multipledose schedule is one in which a primary course of administration may bewith 1–10 separate doses, followed by other doses given at subsequenttime intervals needed to maintain or reinforce the action of thecompositions. Thus, the dosage regimen will also, at least in part, bedetermined based on the particular needs of the subject to be treatedand will be dependent upon the judgement of the reasonably skilledpractitioner.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of “Sized” Glycoconjugates

An exemplary NPr-MenB oligosaccharide-tetanus toxoid conjugate vaccine,hereinafter referred to as CONJ-2, was prepared as follows. The N-acetylgroups of MenB B polysaccharide were removed by heating thepolysaccharide to 110° C. in 2M NaOH for 6 hours in the presence ofNaBH₄. The de-acetylated polysaccharide was exhaustively dialyzed insaturated sodium bicarbonate buffer then stirred with an excess ofpropionic anhydride for 12 hours at ambient temperature. The solutionwas exhaustively dialyzed in water and the N-propionylated meningococcalB (NPr-MenB PS) polysaccharide was recovered by lyophilization.

For preparation of the conjugate vaccine, the NPr-MenB polysaccharidewas partially hydrolyzed in 10 mM sodium acetate at pH 5.5 at 50EC for 2hours. The resulting mixture of oligosaccharides was fractionated onQ-SEPHAROSE (a quaternary ammonium strong anion exchanger).Oligosaccharides having an average degree of polymerization (Dp) of 2–6were first eluted with 100 mM NaCl and discarded. Intermediate-sizedoligosaccharides were eluted with 500 mM NaCl. It was subsequentlydetermined by analytical ion exchange chromatography using a MONO Qcolumn (a quaternary ammonium strong anion exchanger) that theintermediate-sized oligosaccharides ranged in size from Dp 13 to 20(Mean=Dp 13).

A terminal aldehyde group was generated at the non-reducing end of theintermediate-sized oligosaccharides by reacting them with 100 mM sodiumperiodate for 15–30 minutes at ambient temperature in the dark. Excessethylene glycol was used to quench the oxidative reaction and theproduct was desalted on a SEPHADEX G-25 column (a dextran-based column).The oligosaccharide-protein conjugate was prepared by stirring a mixtureof terminal aldehyde containing NPr MenB oligosaccharide with tetanustoxoid (molar ratio of 200:1, respectively) in 0.75 M potassiumphosphate buffer, pH 9.0 with 40 mg/ml of sodium cyanoborohydride forone day at 40EC and two days at ambient temperature. The resultantNPr-MenB oligosaccharide-tetanus toxoid conjugate (CONJ-2) was finallypurified by gel permeation chromatography on SEPHADEX G-100 (adextran-based matrix) using 50 mM sodium phosphate, pH 7.0, 150 mMsodium chloride as the eluting buffer. Sialic acid and proteincompositions of the conjugate vaccine were measured by the Svennerholmresorcinol reaction (Svennerholm, L. (1957) Biochim. Biophys. Acta.24:604) and Lowry assays, respectively. On a weight basis, the finalsaccharide-to-protein ratio of the CONJ-2 conjugates ranged from 0.10 to0.25.

EXAMPLE 2 Characterization of the Glycoconjugates

The CONJ-2 glycoconjugate was characterized as follows. In order todemonstrate covalence (e.g., establishing a covalent linkage between theNPr-MenB OS and the protein carrier), a number of physico-chemicaltechniques can be used, including: SDS-PAGE; Western Blot; SEPHADEXG-100 gel filtration; or the like. For the purposes of the presentstudy, SDS-PAGE was used to establish covalent attachment of theNPR-MenB OS/TT CONJ-2 glycoconjugates by revealing a shift to highermolecular weight for the conjugate band as compared to the carrierprotein band, per Se. Western blot analysis of the CONJ-2glycoconjugates demonstrated covalence by the coincidence of positiveimmunoreactive signals for TT and NPr-MenB PS with specific anti-TT andanti-NPr-MenB PS antisera.

Based on steric factors, the use of oligosaccharides instead of largemolecular weight polysaccharides in the preparation of the CONJ-2glycoconjugates allows for higher coupling efficiency of saccharideantigens onto the protein carrier molecule. The finalsaccharide-to-protein ratio of these NPr-MenB oligosaccharide-basedconjugates range from about 0.10 to 0.25 which corresponds to about 3 to5 NPr-MenB oligosaccharide chains covalently bound per protein carrier.On a per weight basis, the CONJ-2 glycoconjugates appear to have ahigher saccharide loading than a previously reported NPr-MenBpolysaccharide-based conjugate (U.S. Pat. No. 4,727,136) wherein CONJ-2contains, on the average, about 7.5 to 18.8 times more saccharide (using10,000 Daltons as the molecular weight of NPr-MenB PS).

In addition, constructing the CONJ-2 glycoconjugates to havesubstantially homogenous-sized saccharide moieties of a well-definedintermediate chain length (e.g., average Dp of 10–20) is expected toresult in glycoconjugates which display more consistent immunologicalbehavior. Further, the selective end-activation (e.g., selectiveintroduction of the aldehyde group at the non-reducing terminus) of theQ-SEPHAROSE chromatography-purified NPr-MenB oligosaccharides avoids thepossibility of cross-linked, heterogenous structures which could arisefrom the use of NPr-MenB PS molecules with “active” aldehyde groupsintroduced at both termini. In this regard, it is likely thatbi-terminally activated PS (having aldehyde groups at both ends) couldbe derived from a periodate oxidation of N-acylated MenB PS previouslyexposed to NaBH₄ during the N-deacetylation procedure.

EXAMPLE 3 Preparation of Monoclonal Antibodies

4 to 6 week old female CD1 mice were vaccinated by ip injection using acomposition containing an NPr-MenB OS/TT (CONJ-2) glycoconjugate antigenand (except for the last booster injection) FCA. Vaccinations wereadministered at one month intervals for a total of 2 or 3 dosages(including the booster immunization). Three days prior to fusion, theprimed animals were boosted with the NPr-MenB OS/TT (CONJ-2)glycoconjugate antigen in the absence of adjuvant. The final volume ofeach dose was 0.1 ml, which contained 2.5 μg of sialic acid. After thebooster injection, the animals were splenectomized and the spleen cellswere prepared for fusion with myeloma cells.

Approximately one week before fusion, non-secreting murine P3X63-Ag8.653myeloma cells (available from the ATCC under accession numberATCC-1580-CRL), were expanded in complete RPMI-1640 medium with 25 mMHEPES buffer and L-Glutamine (GIBCO BRL 041-02400). The cell cultureswere assessed periodically to monitor cell growth, cell numbers and toscreen for contamination.

On the day of fusion, the spleen cells and the partner P3X63-Ag8.653myeloma cells (AgS cells) were washed, harvested and mixed at a ratio of5:1 (spleen cells:myeloma cells). The cell fusions were performed at 37°C. in the presence of 50% polyethylene glycol (PEG). The resulting cellpellets were harvested and plated into 96 well flat-bottom cell cultureplates (COSTAR 3596) and incubated under suitable conditions (e.g., at37° C. in 5% CO₂). After one day of incubation, selective mediumcontaining hypoxanthine, aminopterin and thymidine (HAT) was added toeach well.

Hybridomas from wells containing growing cells and exhibiting about 10to 25% confluence were selected for screening after about two weeks ofincubation in the HAT selective medium. Selected hybridoma supernatantswere screened using a solid phase avidin-biotinylated NPr-MenB PS basedELISA assay. Specificity of antibody binding in the supernatants wasdetermined using soluble NPr-MenB PS as the inhibitor. Negative controlsincluded RPMI medium, Ag8 myeloma supernatant and irrelevant monoclonalantibody preparations. Pooled polyclonal sera from mice immunized withthe NPr-MenB OS/TT (CONJ-2) glycoconjugate was used as the positivecontrol. After overnight incubation with the supernatants, the reactionwells were washed and bound immunoglobulin was detected with alkalinephosphatase-labelled polyvalent anti-murine immunoglobulins (IgG, IgA,IgM).

Candidate hybridomas were identified based on their demonstrated bindingaffinity for NPr-MenB PS in the above-described ELISA assay. Hybridomassecreting highly reactive antibody molecules were cloned by limitingdilution. Particularly, candidate hybridoma cell lines were plated at0.3, 1.0 and 3.0 cell/well in Terasaki plates (NUNC) in 20 μl ofcloning/expansion medium (Complete RPMI-1640 with IL6). After two weeks,the cultures were visually inspected for growth. Frequency analysis wasperformed using the least squares method described by Lefkovits et al.(1984) Immun. Today 5(9):265. The ELISA assay used to identify reactivesupernatant among the master wells was repeated to assess antibodyactivity on days 7 and 14. Selected clones were then expanded and frozenfor subsequent use in tissue culture and ascites production. A panel of39 hybridomas was thus produced, and the secreted monoclonal antibodymolecules obtained therefrom (termed “SEAM monoclonal antibodies,”particularly, monoclonal antibodies SEAM-1 through SEAM-24, SEAM-26,SEAM-28 through SEAM-31, SEAM-33 through SEAM-36, SEAM-38 throughSEAM-42, and SEAM-48) were prepared for further evaluation.

More particularly, selected monoclonal antibodies were produced eitherin tissue culture, or in ascitic fluid using Pristane-primed 7 to 8 weekold male Balb/c mice. Each animal subject was primed by i.p. injectionwith 0.5 ml Pristane one week prior to inoculation with hybridoma cells.Prior to inoculation, the hybridoma cell concentrations were adjusted tobetween 2.5×10⁶ and 3×10⁶ cells/ml using sterile PBS. The primed animalswere injected i.p. with 1 ml of hybridoma cells, wherein each clonalcell line was inoculated into three different mice. One to two weeksafter inoculation, ascites fluid collection was started and continuedfor a period of approximately one week. The collected fluid wascentrifuged at ambient temperature for 10 minutes at 2700 rpm (1500×g).Supernatants were harvested and pellets discarded. The isolated ascitesfluid was stored at 4° C. over the course of collection, and fluidcollected on different days was pooled, aliquoted and frozen at −70° C.

EXAMPLE 4 Characterization of the Monoclonal Antibodies

The concentrations of unpurified monoclonal antibodies were determinedusing an ELISA capture assay and a radial immunodiffusion assay.Particularly, a capture ELISA procedure was used to determine theconcentration of each of the anti-NPr-MenB PS monoclonal antibodies.Microtiter plates (IMMULON 2, available from Dynatech Laboratories,Inc.) containing 100 μg/well of affinity purified rabbit anti-murineIgG, IgM and IgA (H and L, Zymed) diluted to 1 μg/ml in 10 mM PBS (pH7.4) were incubated overnight at 4EC. After washing three times withPBS, the wells were filled with 250 μl of Blocking Buffer (PBScontaining 1% bovine serum albumin (BSA) and 0.1% sodium azide, pH 7.4)and incubated for 30 to 60 minutes at ambient temperature to blocknonspecific binding sites. The plates were washed three times withWashing Buffer (PBS containing 0.1% TWEEN 20 (polyoxyethylenesorbitanmonolaurate) and 0.1% sodium azide, pH 7.4). Antibodies to be testedwere diluted in Diluting Buffer (PBS containing 1% BSA, 0.1% TWEEN 20(polyoxyethylenesorbitan monolaurate) and 0.1% sodium azide, pH 7.4) andthen added at 100 μl per each well. The plates were covered andincubated overnight at 4EC. Murine IgG1, IgG2b, IgG3 and IgMimmunoglobulin standards (available from Southern BiotechnologyAssociates), at concentrations ranging from 500 ng/ml to 4 ng/ml, wereused to construct standard curves for quantifying antibodyconcentrations.

After incubation overnight, the wells were washed five times with coldWashing Buffer and incubated for 3 hours at 4° C. with 100 μl/well ofalkaline phosphatase conjugated anti-murine IgG, IgM and IgA polyclonalantibodies (H and L, Zymed) that were diluted 1:2000 in Diluting Buffer.The plates were then washed with cold Washing Buffer, and 100 μl offreshly prepared substrate (p-Nitrophenyl phosphate, Sigma) diluted to 1mg/ml in Substrate Buffer (1.0 M diethanolamine, 0.5 mM MgCl₂, pH 9.8)was added to each well. Absorbance values at 405 nm were measured afterapproximately 30 minutes. Immunoglobulin concentrations of themonoclonal antibody preparations were calculated from the standardcurves.

Radial immunodiffusion assays were conducted as follows. Radialimmunodiffusion plates and reagents were obtained from The Binding SiteLimited (Birmingham, England). The assay protocol was then based on themanufacturer's specific instructions supplied with the RID kit. Briefly,calibrator antibody supplied with the kit was reconstituted with anappropriate amount of distilled water. 1:2 and 1:10 dilutions ofcalibrator antibody were prepared. Test samples can be diluted in 1% BSAif necessary. Aliquots of 10 μl (20 μl for IgA and IgG2a subclassantibodies) for calibrator antibody (neat, 1:2, and 1:10 dilutions) andtest samples were applied to separate wells on the plate and incubatedfor 120 hours at room temperature. The concentrations of the antibodieswere determined by measuring the precipitation ring diameters andcomparing these values to a reference table included with the RID kit.

The monoclonal antibodies from tissue culture or ascitic fluid were thenpartially purified as follows. Tissue culture supernatant or ascitescontaining the monoclonals (200 ml or indicated volume) was added slowlyto an equal volume of cold 100% saturated ammonium sulfate (SIGMA, SaintLouis, Mo.) while stirring the solution gently. The monoclonal antibodyand Ammonium sulfate mixture was incubated overnight at 4° C. Thefollowing morning, the mixture was stirred gently to homogeneity andcentrifuged at 5000 rpm in a Sorvall SS34 rotor for 30 minutes at 4° C.After decanting the supernatant, an equal volume of 50% ammonium sulfatesolution (i.e. same volume as the 100% saturated ammonium sulfate) wasused to wash and resuspend the pellet. The resulting mixture wascentrifuged at 5000 rpm in a Sorvall SS34 rotor for 30 minutes at 4° C.The supernatant was then decanted and drained.

For ascites, the pellet was reconstituted in 0.3–0.5 volumes of thestarting volume in PBS Buffer (50 mM sodium phosphate, 150 mM sodiumchloride, pH 7.4).

For tissue culture supernatant, the pellet was reconstituted in 0.1volumes of the starting volume of PBS Buffer. The reconstitutedmonoclonal antibody and ammonium sulfate mixture was placed in adialysis tubing (molecular weight cut off 10,000–12,000) and allowed todialyze in 4 L of PBS overnight. The PBS solution was changed 3 to 4times over the following two days. Monoclonal antibody molecules fromthe dialysis tubes were transferred into a syringe and sterile filteredthrough a 0.2 μm membrane filter, and then stored at −20° C.

The partially purified monoclonal antibody preparations were thencharacterized for (a) immunoglobulin isotype, (b)concentration-dependent binding to NPr-MenB PS, (c) the ability ofvarious NPr-MenB oligomers to inhibit binding to NPr-MenB PS, (d)cross-reactivity with native MenB PS, (e) cross-reactivity with virulentstrains of MenB, (f) complement-mediated bactericidal activity, (g)opsonic activity, and (h) autoreactivity as demonstrated by binding to aneuroblastoma cell line that expresses long chain α2–8 linked polysialicacid at the cell surface. In these experiments, the concentrations ofmonoclonal antibody were measured by the capture ELISA and RID assaydescribed above.

(a) Isotyping of the Antibodies:

The isotypes of the monoclonal antibodies (heavy and light chains) weredetermined by ELISA using the above-described protocol for theanti-NPr-MenB PS ELISA with the only difference that the secondaryalkaline phosphatase-conjugated antibody was specific for IgGsubclasses, IgM, IgA and κ and λ light chains. A kit was also used toisotype the antibody molecules. The kit consisted of typing sticksubstrates coated with goat antibodies specific for the different typesof immunoglobulin peptide chains. The kit provides a peroxidase-labelledspecies specific for anti-murine immunoglobulin to detect the murinemonoclonal antibodies bound to the goat antibodies on the substrate.

As depicted below in Table 1, the isotypic distribution among the 39monoclonal antibodies was found to consist of one IgM and thirty-eightIgG (eight IgG1, five IgG2a, sixteen IgG2b, and nine IgG3). In addition,all antibody molecules had K light chains.

TABLE 1* Binding to Fine SEAM ELISA ELISA Inhibition ELISA EncapsulatedAntigenic Monoclonal Reactivity to of N-Pr-MenB Reactivity to NeisseriaBinding to Opsono- Specificity Antibody Ig N-Pr-MenB Binding by N-Pr-N-Ac-MenB meningitidis CHP134 Bactericidal phagocytotic Group (a) NumberIsotype PS (b) MenB OS (c) PS (d) group B (e) PSA (f) Activity (g)Activity (g) I 10 G1, κ + + + + + + + + + 0 ND 0 11 G2b,κ + + + + + + + + + + + + + ND 18 G2b, κ + + + + + + + + + + + + + + + +20 G2b, κ +/− + + + + 0 0 0 ND 21 G2b, κ +/− + + + + + 0 0 0 ND 26 G2b,κ + + + + + + + + + + + + + ND 28 G2b, κ + + + + + + + + + + + + + + 29G2a, κ + + + + + + + + + + + 0 ND 35 G2b,κ + + + + + + + + + + + + + + + + II 12 G2a, κ + + + +0 + + + + + + + + + 13 G3, κ + + + 0 + + + + + + + + + + + + + 14 G2b,κ + + + + 0 + + + + + + + + ND 15 G2b, κ + + + + 0 + + + + + + + + ND 16G2b, κ + + + 0 + + i + + 0 30 G3, κ + + + 0 + + + + + + + + + + + + +III 1 G3, κ + + 0 0 0 + + ND 3 G2b, κ + + + + + + + 0 + 0 + + + + + + 4G1, κ + + + + 0 i i ND ND 5 G3, κ +/− + 0 + 0 + + + 0 7 G3, κ + + 0 ii + + + 0 8 G3, κ + + + + + + 0 + 0 + + + 0 17 M, κ + + + + 0 0 0 0 ND19 G2a, κ + + + + 0 0 i 0 ND 22 G2b, κ + + + 0 0 i 0 ND 23 G2b, κ + + +0 0 0 0 ND 48 G2b, κ + + + + + + 0 + 0 + + + + IV 2 G3, κ +/− 0 0 +0 + + + 0 6 G3, κ +/− 0 0 0 i 0 ND 9 G1, κ + + 0 0 0 i ND ND 24 G2b,κ + + 0 0 + 0 0 ND ND 31 G1, κ +/− ND + + i ND ND 36 G2a, κ + + +ND + + + + + + + ND 39 G2a, κ +/− ND + + 0 + + 0 ND 40 G1, κ + + + +ND + + + + 0 ND 41 G2b, κ + + ND + + 0 + + 0 33 G1, κ + ND 0 0 0 ND ND34 G3, κ +/− ND 0 0 0 0 ND 38 G1, κ +/− ND 0 0 0 ND ND 42 G1, κ +/− ND0 + i ND ND *The data reported in Table 1 represent the results ofrepeated studies as described herein, and are subject to some variancedue to use of different antigen sources in the ELISA procedure, anddifferent complement sources in the bactericidal assay. (a) Defined bycross-reactivity with N-Ac-MenB PS by ELISA and inhibition ofanti-N-Pr-MenB PS binding by short N-Pr-MenB oligomers. (b)Concentration of monoclonal antibody required to yield an OD of 0.5:+/−, 5–25 μg/ml; +, 1.0–4.9 μg/ml; + +, 0.1–0.9 μg/ml; + + +, 0.01–0.09μg/ml; + + + +, <0.01 μg/ml. (c) 0, <25% inhibition; +, 26–48%inhibition; + +, 49–74% inhibition; + + +, 75–100% inhibition whentested at OD 0.5 to 1; Dp 3.8 N-Pr-MenB fragments. (d) 0, OD < 0.15; +,OD 0.15–0.5; + +, OD 0.5–1.0; + + +, OD > 1.0 when tested at 5 to 25μg/ml of antibody by ELISA. (e) 0, no detectable binding to encapsulatedstrains when tested at 100 μg/ml; +, binding to encapsulated strains8047 and NmB, but not to non-encapsulated strain M7; i, indeterminate(see text). (f) 0, no binding activity to polysialic acid (PSA) whentested at 100 μg/ml of antibody; + +, binding activity when tested at 10μg/ml and inhibitable by neuraminadase treatment; +, binding activitydetected at 100 but not 10 μg/ml; i, indeterminate is binding activitynot inhibitable by neuraminadase treatment. (g) + + + +, activity withboth rabbit and human complement, and in the absence of complement; + ++, activity with both rabbit and human complement; + +, activity withrabbit complement, no activity with human complement; 0, no activitywith rabbit complement or human complement (also includes antibodiesonly tested with rabbit complement); ND, not done.

(b) Concentration-Dependent Binding to NPr-MenB PS:

A solid phase ELISA procedure was used to assess the concentrationdependent binding of the antibody molecules to NPr-MenB PS in thepresence of buffer alone or 25 μg/ml of a soluble NPr-MenB PS inhibitor.Biotinylated NIPr-MenB PS-ADH was prepared using the method of Sutton etal. (1985) J. Immunol. Methods 82:215. Microtiter plates (IMMULON 2,available from Dynatech Laboratories, Inc.) containing 100 μl/well ofavidin (4 μg/ml Extr Avidin, Sigma) in 10 mM PBS (pH 7.4) were incubatedovernight at 4EC. After washing three times with PBS, 100 μl ofbiotinylated NPr-MenB PS in PBS was added to each well and incubated at37EC for 2 hours. The plates were washed three times with PBS, and thewells were filled with 250 μl of Blocking Buffer and incubated for 30 to60 minutes at ambient temperature to block nonspecific binding sites.

After blocking, the plates were washed three times with Washing Buffer.50 μl aliquots of various dilutions of the monoclonals were added towells of replicate plates containing either 50 μl of Diluting Buffer or50 μl of Diluting Buffer containing 50 μg of soluble NPr-MenB PS per ml(for a final inhibitor concentration of 25 μg/ml). The plates were thencovered and incubated overnight at 4° C. On the following day, the wellswere washed five times with cold Washing Buffer and then incubated for 3hours at 4° C. with 100 μl/well of alkaline phosphatase conjugatedanti-murine IgG, IgM and IgA polyclonal antibodies (Zymed) diluted1:2000 in Diluting Buffer. The plates were then washed with cold WashingBuffer, and 100 μl of freshly prepared substrate (p-Nitrophenylphosphate, Sigma) diluted to 1 mg/ml in Substrate Buffer was added toeach well. Absorbance values at 405 nm were measured after approximately30 minutes.

FIGS. 1A–1D show the dose-response binding activity of fourrepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-5,SEAM-16 and SEAM-18, respectively), to solid phase NPr-MenB PS asdetermined by ELISA. Data shown are for the antibodies diluted in buffer(●), or in buffer containing 25μg/ml of soluble NPr-MenB PS (∘).Different ranges for the X axis in the data are used, wherein monoclonalantibodies SEAM-3, SEAM-16 and SEAM-18 are shown at 0.0001 to 1 μg/ml,and monoclonal antibody SEAM-5 is shown at 0.1 to 100 μg/ml. Theconcentration of antibody sufficient to yield an OD of 0.5 afterincubation with substrate varied considerably (compare binding of SEAM-5to binding of SEAM-18).

Table 1 summarizes the respective concentration ranges of antibodyrequired to yield an OD of 0.5 in an ELISA for each of the 39 SEAMmonoclonal antibodies. The most likely explanation for the largeheterogeneity in the values shown is differences in antibody avidity toNPr-MenB PS.

(c) Inhibition of Antibody Binding to NPr-MenB PS by Oligomers:

A competitive solid phase ELISA procedure was used to assess the abilityof NPr-MenB oligomer inhibitors to inhibit binding of the monoclonalantibody molecules to solid phase NPr-MenB PS. The assay was performedas described above for the anti-NPr-MenB PS ELISA with the exceptionthat the monoclonal antibodies were pre-diluted to concentrations toyield an OD of 0.5 to 1. The monoclonal antibodies were added to wellsof replica plates, each containing one of the following solubleinhibitors to yield a final inhibitor concentration of 25 μg/ml: highmolecular weight (HMW) NPr-MenB PS; or low molecular weight (LMW)NPr-MenB OS (having an average Dp of 3.8).

The plates were covered and incubated overnight at 4° C. On thefollowing day, the wells were washed five times with cold Washing Bufferand then incubated for 3 hours at 4° C. with 100 μl/well of alkalinephosphatase conjugated anti-murine IgG, IgM and IgA polyclonalantibodies (Zymed) diluted 1:2000 in Diluting Buffer. The plates werethen washed with cold Washing Buffer, and 100 μl of freshly preparedsubstrate (p-Nitrophenyl phosphate, Sigma) diluted to 1 mg/ml inSubstrate Buffer was added to each well. Absorbance values at 405 nmwere measured after approximately 30 minutes. Percent inhibition wascalculated as compared to binding in the absence of inhibitor.

FIG. 2 depicts the inhibition of binding of four representativeanti-NPr-MenB PS monoclonal antibodies (SEAM-2, SEAM-3, SEAM-16 andSEAM-18) to solid phase NPr-MenB PS by either 25 μg/ml of soluble highmolecular weight (HMW) NPr-MenB PS inhibitor (▪), or 25 μg/ml of lowmolecular weight (LMW) NPr-MenB oligosaccharide (average Dp of 3.8)inhibitor (□).

The HMW NPr-MenB PS inhibitor provided approximately 75% to 95%inhibition in all monoclonal antibodies tested. Differences in fineantigenic specificity in the monoclonal antibodies are evident from thedifferent respective patterns of inhibition with the LMW inhibitortested. For example, binding of SEAM-3 and SEAM-18 to NPr-MenB PS iscompletely inhibited by the soluble LMW inhibitor of NPr-MenB PS. Incontrast, SEAM-2 and SEAM-16 are not significantly inhibited by theoligomers (less than 20%). The results of LMW NPr-MenB OS inhibition forall of the monoclonal antibodies are depicted in Table 1. In addition,as described below, other differences in the fine antigenic specificityof the monoclonals are evident by the differences observed incross-reactivity to NAc-MenB PS in ELISA and differences in binding tohost polysialic acid.

(d) Cross-Reactivity with NAc-MenB PS:

The monoclonal antibodies were evaluated for their ability tocross-react with the NAc-MenB polysaccharide as demonstrated by directbinding to NAc-MenB PS in a solid phase ELISA format. The method usedwas similar to that described above for the NPr-MenB PS ELISA, with theexception that NAc-MenB PS-ADH was used as the solid phase antigeninstead of biotinylated NPr-MenB PS.

50 μl aliquots of various dilutions of the monoclonals were added towells of replicate plates containing either 50 μl of Diluting Buffer or50 μl of Diluting Buffer containing 50 μg of soluble NAc-MenB PS per ml(for a final inhibitor concentration of 25 μg/ml). The plates were thencovered and incubated overnight at 4° C. On the following day, the wellswere washed five times with cold Washing Buffer and then incubated for 3hours at 4° C. with 100 μl/well of alkaline phosphatase conjugatedanti-murine IgG, IgM and IgA polyclonal antibodies (ZYMED) diluted1:2000 in Diluting Buffer. The plates were then washed with cold WashingBuffer, and 100 μl of freshly prepared substrate (p-Nitrophenylphosphate, SIGMA) diluted to 1 mg/ml in Substrate Buffer was added toeach well. Absorbance values at 405 nm were measured after approximately30 minutes.

FIG. 3 depicts the binding of five representative anti-NPr-MenB PSmonoclonal antibodies (SEAM-12, SEAM-16, SEAM-18, SEAM-2, and SEAM-3) tothe solid phase NAc-MenB PS. As can be seen, three of the antibodies,SEAM-12, SEAM-16 and SEAM-18, showed significant binding when tested at0.5 and/or 5 μg/ml of antibody. Two other antibodies, SEAM-2 and SEAM-3,previously shown to be negative in a screening assay, were confirmed asnegative when tested at 5-fold higher concentrations (25 μg/ml ofantibody). The cross-reactivity of each of the 39 monoclonal antibodieswith the NAc-MenB PS was scored over a range of (+++) for highly crossreactive, to (0) for non cross-reactive. The results are depicted inTable 1. As can be seen, sixteen of the monoclonal antibodiescross-reacted with the NAc-MenB PS, and four minimally cross reacted (±)(FIG. 1). Specificity of the cross-reactivity of these twenty positive,or weakly positive monoclonal preparations was confirmed by inhibitionof binding using soluble NAc-MenB PS. The 26 non cross-reactivemonoclonal antibodies showed no significant binding to solid phaseNAc-MenB PS when tested at antibody concentrations up to 25 μg/ml.

(e) Bacterial Binding Assay:

The ability of the anti-N-Pr meningococcal B polysaccharide antibodiesto bind to the surface of pathogenic strains of N. meningitidis Group Bwas determined using flow cytometric detection of indirectimmunofluorescence assay. Two fully encapsulated meningococcal B testorganisms were used, strain 8047 (the strain used to measurebactericidal activity, see below) and NmB. A third unencapsulatedstrain, M7, which is a transposon-containing mutant of NmB (Stephens etal. (1991) Infect. & Immun. 59:4097–4102) was used as a negative controlfor specificity of antibody binding to the capsular polysaccharide.Bacterial cells grown to mid-log phase in Mueller-Hinton broth and 0.25%glucose were harvested and resuspended in Blocking Buffer at a densityof ˜10⁶ cells per ml. The monoclonal antibodies (concentration of 10 or100 μg/ml) were then added and allowed to bind to the cells on ice for 2hours. Following two washes with Blocking Buffer, the cells wereincubated with FITC-conjugated F(ab′)₂ fragment goat anti-mouse IgG(H+L) (Jackson Immune Research, West Grove, Pa.), fixed with 0.25%formaldehyde in PBS buffer, and analyzed by flow cytometry.

Positive control antibodies included meningococcal-specific serotypingand subtyping monoclonal antibodies (MN2C3B, MN16C13F4, RIVM, Bilthoven,the Netherlands). The negative control consisted of a mouse IgGmonoclonal antibody of irrelevant specificity.

FIGS. 4A–4G show the results from a representative experiment.Monoclonal antibodies SEAM-3 and SEAM-18 show strong capsular-specificbinding to both encapsulated test strains. (FIGS. 4C and 4D,respectively) in this indirect fluorescence flow cytometry assay. Incontrast, monoclonal antibodies SEAM-9 and SEAM-10 were negative in thisassay (FIGS. 4E and 4F). As summarized in Table 1, twenty-four of theanti-N-Pr meningococcal B polysaccharide antibodies showed evidence ofbacterial binding when tested at 100 μg/ml. Two additional antibodiesshowed evidence of minimal binding to both encapsulated andnon-encapsulated mutant strains. Bacterial binding of these antibodieswas scored as indeterminant (i). See, for example, the binding of SEAM-7depicted in FIG. 4G.

(f) Complement-Mediated Bactericidal Activity:

A bactericidal assay was conducted using the methods described byMandrell et al. (1995) J. Infec. Dis. 172:1279, with the followingmodifications: the organism was grown in Mueller-Hinton broth containing0.25% glucose; and serum diluting buffer consisted of Gey's bufferinstead of barbitol buffer. In several experiments, different sources ofcomplement were used: these included two different infant rabbit serumpools (referred to as Rab C I and Rab C II) and human agammaglobulinemicserum (referred to as Hu C).

The percent survival of N. meningiditis strain 8047 when incubated withdifferent concentrations of antibody and 20% complement is shown forfour representative monoclonal antibodies (FIGS. 5A–5D). Each antibodyshown was tested with three different complement sources: infant rabbitserum pool I (▴), infant rabbit serum pool II (●), and humanagammaglobulinemia (∘). For SEAM-5 and SEAM-12, a similar dose responsefor each antibody was observed for each of the three complement sources.In contrast, SEAM-18 required higher antibody concentrations to elicitbacterial killing in the presence of human complement than were requiredwith either source of rabbit complement. SEAM-3 showed effective killingwhen tested with the two rabbit complement sources, and no activity withthe human complement source. The ability of each of the monoclonalantibodies to activate complement-mediated bacterial lysis is reportedin Table 1. There are examples of bactericidal antibodies that crossreact with NAc-MenB PS by ELISA (e.g., SEAM-18, SEAM-30, and SEAM-35).There also are examples of bactericidal antibodies that show nocross-reactivity with NAc-MenB PS (e.g., SEAM-2, SEAM-5, SEAM-7, andSEAM-8).

(g) Opsonic Activity:

Opsonic activity of the monoclonal antibodies can be measured by avariety of established methods. Sjursen et al. (1987) Acta Path.Microbiol. Immunol. Scand., Sec. C 95:283, Halstensen et al. (1989)Scand. J. Infect. Dis. 21:267, Lehmann et al. (1991) APMIS 99:769,Halstensen et al. (1991) NIPH Annals 14:157, Fredlund et al. (1992)APMIS 100:449, Guttormsen et al. (1992) Infect. Immun. 60:2777,Guttormsen et al. (1993) J. Infec. Dis. 167:1314, Bjerknes et al. (1995)Infect. Immun. 63:160, and Hayrinen et al. (1995) J. Infect. Dis.171:1481.

In one opsonization assay, N. meningitidis freshly grown on GN agarplates (Greiner Labortechniek, Greiner BV, Alphen a/d Rijn, Netherlands)at 37° C. was used to inoculate 8 ml of Mueller Hinton broth (Difco,Detroit, Mich.) to obtain an initial OD of 0.1. The bacteria were grownto log phase (660 nm absorbance of 0.75–0.85) with vigorous shaking. Thecells were transferred to sterile plastic tubes with caps andcentrifuged for 10 minutes at 3500 rpm.

Cells were fixed by adding 4 ml of 70% ethanol and incubating for atleast 1 hour 4° C. The fixed cells were again pelleted by centrifugationfor 10 minutes at 3500 rpm and resuspended in sterile phosphate bufferedsaline (PBS) to yield an OD of 1.0. The cell suspension (1.35 ml) wasadded to an eppendorf tube and centrifuged for 5 minutes at 10,000 rpm.The supernatant was discarded, and another 1.35 ml was added to the sametube followed by centrifugation to yield 1×10⁹ cells per tube. A 1.0mg/ml solution of fluorescein isothiocyanate (FITC) in PBS (Sigma, St.Louis, Mo.) was prepared and sonicated for 5 minutes, then centrifugedfor 5 minutes at 10,000 rpm. The FITC-PBS solution (50 μl) was added toeach tube of bacteria and then incubated for 1 hour at 37° C. withslight agitation. PBS (950 μl) was added to each tube and centrifugedfor 2 minutes at 10,000 rpm. The pellet was washed once with 1 ml of PBSand once with 1 ml of BSA-Hanks balanced salt solution (BSA-HBBS). TheFITC labelled meningococci were reconstituted in 1% BSA-HBBS and dividedinto 100 μl aliquots which were stored at −20° C. until use in theassay.

Human polymorphic nuclear cells (PMN) were isolated from the peripheralblood of healthy adults in heparin-containing tubes (Becton Dickinson,Mountain View, Calif.). A volume of 10 ml of blood was diluted with anequal amount of phosphate buffered saline (PBS; pH 7.4) and layered on aFicoll histopaque gradient consisting of 10 ml of Ficoll Paque™(Pharmacia, Uppsaila, Sweden) on top of 12 ml of histopaque (density1.119, Sigma Diagnostics, St. Louis, Mo.). After centrifugation at 400×gfor 20 minutes at room temperature, the PMN were collected from theupper part of the histopaque and ice cold RPMI medium (Roswell ParkMemorial Institute, NY) containing 1% gelatin was added. Cells werecentrifuged at 250×g and the residual erythrocytes were lysed byresuspending the cells in 9 ml of ice cold distilled water. After 1minute, concentrated PBS and RPMI-gelatin was added to make the cellsuspension isotonic. The PMN were centrifuged and resuspended in RPMImedium to a density of 1×10⁷/ml. The purity and viability of the PMN wasgreater than 95%.

To a microtiter plate was added appropriate dilutions of monoclonalantibody to be tested (diluted in BSA-HBBS), 5 μl of 10% humancomplement (in BSA-HBBS), and 25 μl of FITC-labelled bacteria suspensionto yield a total volume of 50 μl. Selected antibodies were testedwithout complement, and with up to three different complement sources:normal pooled human serum; agammaglobulinemic serum; and infant rabbitserum, varying the complement concentration from 1 to 10%. Each assayincluded a positive and negative antibody control, as well as acomplement, non-opsonization and a cells-only control. The opsonizationreaction was allowed to proceed for 30 minutes at 37° C. on a shakerbefore terminating the reaction by placing the microtiter plate on ice.

Phagocyte cell suspension (50 μl) was added to a final concentration of5×10⁶ cells/ml. This gives a ratio of bacteria to phagocytes of 10:1.Phagocytosis was allowed to proceed for 30 minutes at 37° C. on ashaker, after which time it was placed on ice. Cold BSA-HBBS (100 μl)was added to each well. The plates were centrifuged for 10 minutes at1100 rpm. Supernatants were aspirated from the wells and the cells werewashed twice more with 150 μl of cold BSA-HBBS. Cold BSA-HBBS (150 μl)was then added, and the resulting cell suspensions were transferred tosterile tubes. A solution of 2% paraformaldehyde (Polysciences, Inc.,Warrington, Pa.) in PBS was added to fix the cells. The samples werethen analyzed by indirect florescence flow cytometry.

The results of the opsonization experiments for sixteen representativeSEAM monoclonal antibodies are reported in Table 1. All antibodies foundto be opsonic were also bactericidal in the assay described above usingat least one of the complement sources. However, as can be seen in Table1, there are examples of antibodies that were bactericidal but notopsonic (see, e.g., SEAM-2, SEAM-5, SEAM-7, SEAM-16, and SEAM-41).

(h) Evaluation of Autoreactivity:

Partially purified tissue culture supernatants containing the 39 SEAMmonoclonal antibodies were evaluated for autoreactivity to hostpolysialic acid. In one assay, the monoclonal antibodies were assessedfor their ability to cross-react with the human neuroblastoma cell lineCHP-134 (Livingston et al. (1988) J. Biol. Chem. 263:9443) using flowcytometric detection of indirect immunofluorescence. In this assay, theCHP-134 cells, which express long chain polysialic acid (PSA) associatedwith neuronal cell adhesion molecule (NCAM) on their surface, serve ascellular markers for human PSA antigens. In control experiments, nearlyconfluent cell cultures were collected in 50 ml centrifuge tubes andcentrifuged at 1000×g. After the supernatant was decanted, 5 ml ofBlocking Buffer was added to resuspend the cells. The cells were thencounted in a hemacytometer, and divided into two equal aliquots. Onealiquot was incubated for 2 hours at ambient temperature withexoneuraminidase (10 units/10⁸ cells, SIGMA Chemical Co., Saint Louis,Mo.); the other aliquot was treated identically but without enzyme.After incubation, the cells from each aliquot were distributed amongindividual reaction tubes so that each tube contained 10⁶ cells. To washthe cells, 2 ml of Blocking Buffer was added to each reaction tube, thetubes centrifuged at 1000 rpm in a Sorvall RT-600B for 6 minutes at 20°C., and the supernatant aspirated off. The washed cells were incubatedfor 2 hours in a total volume of 200 μl on ice with either no antibody,or the indicated concentration (usually 10 or 100 μg/ml) of the testantibody (i.e., SEAM MAbs).

Control antibodies in the assay included: (1) an IgG monoclonal antibodyof irrelevant specificity (VIIG10, as a negative control); (2) an IgManti-polysialic acid monoclonal antibody (2-1B, as a positive control);and (3) an anti-CD56 monoclonal antibody specific for the proteinbackbone of NCAM (Immunotech, Marseille, France). Blocking Buffer (2 ml)was added to each reaction tube, and the tubes were centrifuged at 1000rpm in the SORVALL RT-600B for 6 minutes at 20° C. Followingcentrifugation, the supernatant was aspirated off and the cellsincubated for 1 hour at ambient temperature with 150 μl of fluoresceinisothiocyanate (FITC)-conjugated F(ab′)₂ fragment goat anti-mouse IgG(H+L) (diluted to 4 μg/ml) (Jackson Immune Research, West Grove, Pa).After washing with Blocking Buffer, 400 μl of 0.25% formaldehyde in PBSbuffer (50 mM sodium phosphate, pH 7.0, 150 mM sodium chloride) wasadded to the cells, and the cells were analyzed by flow cytometry usinga FACSCAN™ cell sorter (Becton-Dickinson, Mountain View, Calif.).

All antibodies were tested at final concentrations of 10 and 100 μg/mlof antibody in replicate, using untreated cells, and cells that had beenpre-treated with neuraminidase. This treatment cleaves the surfacepolysialic acid and provides a control in the assay for specificity ofantibody binding to polysialic acid. In a typical experiment (FIGS.6A–6I), cells incubated without primary antibody, or with a controlmonoclonal antibody having an irrelevant antigenic specificity, showvery little fluorescence (approximately 98% of the cells have <10 unitsof fluorescence, FIG. 6A). In contrast, virtually all cells treated withthe anti-NAc MenB PS monoclonal antibody, 2-1B, fluoresce strongly (FIG.6B, left). This fluorescence is decreased to control levels when theantibody is incubated with cells that had been pre-treated withneuraminidase (FIG. 6B, right). Similarly, cells treated with anti-CD56fluoresce strongly (FIG. 6C). With this antibody, the fluorescence isunaffected by pre-treatment of the cells with neuraminidase since theCD56 determinant is located in the protein backbone of NCAM and isunaffected by the removal of polysialic acid with neuraminidase.

The SEAM-5 antibody gives no detectable binding when tested at 100 μg/ml(FIG. 6D), and is considered as negative in this assay. The SEAM-35antibody shows strong polysialic acid-specific binding when tested at 10or 100 μg/ml (FIGS. 6E and 6F), and is considered positive. A fewanti-NPr MenB PS monoclonal antibodies show binding when tested at 100μg/ml, but appear to be negative when tested at 10 μg/ml (see, e.g.,SEAM-12 in FIGS. 6G and 6H). Such antibodies are considered minimallyautoreactive for the purposes of this application. A rare antibodyappeared to have weak reactivity with the neuroblastoma cell line thatwas unaffected by the by pre-treatment of the cells with neuraminidase(see SEAM-7, FIG. 6I). The autoreactivity of such antibodies withpolysialic acid was scored as indeterminant in the assay, and theseantibodies were also considered to have minimal autoreactivity to hostPSA for purposes of this application.

Table 1 summarizes the autoantibody activity of each antibody asdetermined in this indirect fluorescence flow cytometry assay.Cross-reactivity with polysialic acid antigens expressed in CHP-134cells was closely correlated with the cross-reactivity of the antibodieswith NAc-MenB PS in the ELISA assay. As shown in Table 1, monoclonalantibodies that did not cross react with NAc-MenB PS in the ELISA alsodid not bind to CHP-134 cells, while all of the antibodies thatcross-reacted with NAc-MenB PS in the ELISA also cross-reacted with PSA.This correlation between the two assays was not unexpected since thepolysaccharide covalent structure of NAc-MenB PS and the host PSA isreported to be the same.

EXAMPLE 5 Passive Immunization Using SEAM Monoclonal AntibodyCompositions

In order to assess the ability of the above-characterized SEAMmonoclonal antibodies to provide passive protection against bacterialchallenge, the following immunization study was carried out.

Animals: Outbred infant SPF (specific pathogen-free) albino Wistar ratswere obtained from the Helsinki University Animal Center (Helsinki,Finland).

Bacterial Strains: Neisseria meningitidis group B strain IH 5341, ahuman patient isolate with MenB:15:p1.7, 16 phenotype, plus 1 to 2additional other group B bacterial strains (e.g. M355; B:15:P1.15) wereused. All bacteria strains were rat passaged five times and stored inskim milk at −70° C. For each experiment, a fresh inoculum was takenfrom the stock and cultivated on gonococcal (GC) medium base (GC-agar IIBase, Becton Dickinson, Mountain View, Calif.) supplemented withISOVITALEX, L-tryptophan and hemoglobin. After incubation overnight at37° C. in 5% CO₂, several colonies were inoculated into a culture flaskcontaining 20 ml of brain-heart infusion broth and incubated at 37° C.in a rotatory shaker at 150 rpm until the optical density (Klett 90)corresponded to 10⁸ cfu/ml. The cultures were then diluted in phosphatebuffered saline (PBS) corresponding to 10⁶ cfu/ml for use. The actualnumber of viable bacteria in a challenge dose was determined by countingthe cfu after serial dilution of the suspension in PBS and plating onproteose peptone agar.

Immunizations: In each experiment 3–4 litters of 4–6 day old infant ratswere randomly selected and divided into experimental groups of 6 animalseach and injected intraperitoneally with either a SEAM monoclonalantibody composition (in 0.9% saline), saline solution (0.9%), orcontrol antibodies. In each group, three animals were inoculated withthe SEAM antibodies (at doses of 0.4 μg, 2 μg, and 10 μg, respectively),two animals were used as negative controls (one received injection withsaline alone while the other received injection with a monoclonalantibody of irrelevant specificity), and a positive animal received aninjection of an anti-Men B polysaccharide antibody.

Bacterial Challenge: One to two hours after the initial injection, theinfant rats received a bacterial challenge injection intraperitoneallyof 10⁵ Neisseria meningitidis group B bacteria of the strain IH 534 (ratpassaged five times) in a final volume of 100 μl. Six hours afterbacterial inoculation, bacteremia and meningitis development wasassessed by culturing blood and cerebrospinal samples taken from theinfant rats.

The results of the study (protection from N. meningitidis bacteremia)for six representative SEAM monoclonal antibodies (SEAM-5, SEAM-7,SEAM-8, SEAM-10, SEAM-12, and SEAM-18) are depicted below in Table 4. Ascan be seen, the SEAM-12 and SEAM-18 antibodies are strongly protective,the SEAM-7 and SEAM-8 antibodies partially protective, with the SEAM-5and SEAM-10 antibodies providing no protection up to a dose of 10μg/pup.

TABLE 2 Blood Titer in cfu/ml × 10⁵ (% of Cerebral Blood negative SpinalFluid SEAM Mab (positives/all) control) (positives/all) Dose: 10 μg/pup 5 5/6  0.63 (31%) 3/6  7 0/6 <0.01 (<1%) 0/6  8 1/6 <0.01 (<1%) 0/6 106/6 10.67 4/6 (>100%) 12 0/6 <0.01 (<1%) 0/6 18 0/6 <0.01% (<1%)    0/6Dose: 2 μg/pup  5 6/6  0.37 (18%) 2/6  7 4/6  0.04 (<1%) 0/6  8 6/6  2.52 (>100%) 4/6 10 6/6 10.35 5/6 (>100%) 12 1/6  0.01 (<1%) 1/6 181/6 <0.01% (<1%)    1/6 Dose: 0.4 μg/pup  5 5/5   5.65 (>100%) 4/5  76/6   9.28 (>100%) 5/6  8 6/6  1.50 (63%) 4/6 10 6/6 10.67 4/6 (>100%)12 6/6  9.51 (76%) 5/6 18 5/5  3.51% 3/5 (>100%)

EXAMPLE 6 Identification of Peptide Mimetics of MenB Antigen Using SEAMMonoclonal Antibodies

The following procedures were carried out in order to identify peptidemimetics that interact with the SEAM monoclonal antibodies of thepresent invention. Phage display peptide libraries were constructed inan M13 vector using techniques known to those skilled in the art. Adeyet al. (1996) “Construction of Random Peptide Libraries in BacteriophageM13,” in Phage Display of Peptides and Proteins, Kay et al., eds.,Academic Press, San Diego, Calif. Particularly, linear 8mers (L8),cyclic 6mers (C6) and single C (C1) peptides were displayed asN-terminal extensions of the pIII bacteriophage protein. Thecharacteristics of the libraries are presented below in Table 3.

TABLE 3^(a) Number of Library Randomized Segment^(b,c) Sequences Linear8mer A E X X X X X X X X G G 2.5 × 10¹⁰ (L8) (P)_(6 . . .) Cyclic 6mer AE C X X X X X X C 6.4 × 10⁷  (C6) (P)_(4 . . .) Single C (C1) A E X X XX X X X X G C 2.5 × 10¹⁰ (P)_(6 . . .) ^(a)Peptides are displayed asfusions with M13 phage protein, pIII. ^(b)X represents a random aminoacid, all other are standard single letter code. ^(c)(P)₄ or (P)₆ refersto either four or six Proline residues, respectively.

Panning of the libraries was carried out using the techniques describedby Smith et al. (1993) Methods in Enzymology 217:228, with the exceptionthat the antibodies were absorbed directly to microtiter plates. 100 μlsolutions containing representative monoclonal antibodies (1 μg/ml ofSEAM-2, SEAM-3, SEAM-5, SEAM-7, SEAM-12, SEAM-16, SEAM-18, and SEAM-28),or a corresponding concentration of control antibodies (a murineanti-MenB PS-specific monoclonal (2-1B), a human anti-Hib PS monoclonal(ED8), and a murine monoclonal of irrelevant specificity (Laz2))wereincubated overnight at 4EC in microtiter plates (IMMULON II). Afterwashing the wells with PBS, Blocking Solution (5% (w/v) non-fat drymilk, 0.2% (w/v) TWEEN 20 (polyoxyethylenesorbitan monolaurate), 0.02%(w/v) sodium azide in PBS) was added to completely fill the wells, andthe plates were then incubated at ambient temperature for 3 hours. Theblocked plates were washed six times with PBS.

Approximately 10¹⁰ pfu of phage were added to triplicate wells in atotal volume of 100 μl per well. The plates were incubated with thephage overnight at 40° C. Each well was then washed nine times with PBS,and the bound phage released by adding to each well 100 μl of 0.2 Mglycine, HCl (pH 2.2) buffer and incubating at ambient temperature for 1hour. The buffer solutions from respective triplicate wells werecombined, and the pH adjusted to 8 by addition of 20 μl 1.5 M Tris (pH8.8) buffer per 100 μl of solution. A freshly grown culture (2 ml) of E.coli (XL1-Blue) at a density of OD_(550nm)=0.4–0.6 in LB mediacontaining 0.2% (w/v) maltose and 12 μg/ml tetracycline (LB-mal, tetmedia) was added to the combined solutions of released phage. The cellsand phage were incubated at 37° C. for 20 minutes, after which 20 ml ofmedia was added. The cells were grown overnight at 37° C., then pelletedby centrifugation (5000×g for 10 minutes). The supernatant was filteredthrough a 0.2 μm membrane, and the phage precipitated by adding 0.15volumes of 20% (w/v) polyethylene glycol 8000, 4 M NaCl., and allowingthe mixture to stand at 4° C. overnight. Precipitated phage werecollected by centrifugation (10,000×g for 10 minutes), and thenresuspended in 20 ml PBS (approximately 10¹² pfu/ml).

Each panning was repeated 3 or 4 times for each screen. Finally, phagereleased from the final pan were used to infect XL1-Blue cells andseveral serial dilutions were plated directly on LB-agar plates.Individual plaques were selected and amplified in 5 ml cultures ofXL1-Blue (LB-mal, tet media). DNA from the phage was prepared usingQIA8-Prep™ columns (Quiagen) and sequenced using a Sequenase™ kit(Amersham) according to the manufacturer's instructions.

A total of 67 unique peptide sequences (Peptides Pep 1–Pep 67) wereselected by the SEAM monoclonal antibodies. These peptide sequences aredepicted in FIG. 7 as SEQ ID NOs. 1–67. Of these sequences, 13 wereidentified on more than occasion (Table 4). With one exception, none ofthe sequences selected by the control antibodies (2-1B, ED8 and Laz2)were identical or significantly homologous to those selected by the SEAMmonoclonal antibodies. The single exception (SEQ ID NO. 9) was selectedby both SEAM-3 and the Laz2 control antibody. However, this result waspossibly due to a cross-contamination between reagents since bothexperiments were conducted at the same time.

TABLE 4 Number of Identical Antibody Peptide Sequence Isolates SEAM-2 Pep 10 3 (SEQ ID NO. 10) SEAM-2  Pep 13 2 (SEQ ID NO. 13) SEAM-2  Pep 142 (SEQ ID NO. 14) SEAM-3, 16, 18 Pep 1  37  (SEQ ID NO. 1) SEAM-5  Pep2  3 (SEQ ID NO. 2) SEAM-7  Pep 3  5 (SEQ ID NO. 3) SEAM-7, 18 Pep 4  2(SEQ ID NO. 4) SEAM-7, 18 Pep 5  2 (SEQ ID NO. 8) SEAM-12 Pep 6  4 (SEQID NO. 6) SEAM-18 Pep 7  3 (SEQ ID NO. 7) SEAM-18 Pep 12 4 (SEQ ID NO.12) SEAM-28 Pep 8  2 (SEQ ID NO. 8) SEAM-28 Pep 67 2 (SEQ ID NO. 67)

EXAMPLE 7 Characterization of the Peptide Mimetics

For characterization of the antibody binding to synthetic peptides, thepartially purified monoclonal antibodies were purified further on aBIOCAD® perfusion chromatography workstation using a Poros G/M protein Gcolumn (4.6mm×100 mm) with a column volume of 1.7 ml (PerSeptiveBiosystems, Framingham, Mass.). The protein G column was equilibratedwith 10 column volumes of PBS buffer. Monoclonal antibody preparations(2 ml) from either ascites or tissue culture resuspended in PBS wereinjected onto the protein G column. After washing with 5 column volumesof PBS buffer, monoclonal antibody was eluted from protein G column witha 0.2 M Glycine-HCl, 150 mM sodium chloride (pH 2.5) buffer. The elutedantibodies were monitored with internally equipped spectrophotometricdetectors at both 220 nm and 280 nm, and the elution peak collected andstored at 4° C. The pH of each 1 ml fraction was raised to 8.0 by adding100 μl of 1.5 M Tris (pH 8.8) immediately upon collection.Concentrations of the purified monoclonal antibodies were determinedwith a spectrophotometer from absorbance at 280 nm using an extinctioncoefficient of 0.71 mg⁻¹ ml cm⁻¹.

An ELISA was used to determine the ability of anti-NPr-MenB PSantibodies to recognize synthetic peptides corresponding to selectedpeptide mimetic sequences identified in Table 4. Synthetic peptides werepurchased from Biosynthesis (Lewisville, Tex.). To facilitate absorptionto the ELISA plate, the peptides were modified by the addition at theamino terminus of a hydrophobic tail (Lauryl-GLY-GLY). Further, thepeptides were carboxyl-terminal amides. The synthetic peptides (1 mg)were resuspended in 100 μl of dimethyl sulfoxide (Sigma, St. Louis, Mo.)and an aliquot was then diluted further in 50 mM Hepes (FisherScientific, Pittsburgh, Pa.) pH 8.0, 150 mM NaCl (Sigma, St. Louis, Mo.)and 0.02% sodium azide (Sigma, St. Louis, Mo.) to a peptideconcentration of 10 μg/ml. Microtiter plates (Immulon 2®; DynatechLaboratories Inc., Chantilly, Va.) containing 100 μl/well of a 10 μg/mlpeptide solution in 50 mM Hepes buffer were incubated overnight at 4° C.After washing the plates 3 times with phosphate buffered saline (PBS, pH7.4), the wells were filled with 200 μl of Blocking Buffer and incubatedfor 1–2 hours at room temperature to block non-specific binding sites.The plates were then washed 5 times with Washing Buffer.

Various dilutions of the SEAM monoclonal antibodies (50 μl) to be testedfor peptide binding were added to duplicate plates containing either 50μl of Diluting Buffer or 50 μl of Diluting Buffer containing 50 μg ofsoluble NPr-MenB PS per ml (final inhibitor concentration of 25 μg/ml).The plates were then covered and incubated overnight at 4° C. Thefollowing day plates were washed 5 times with Washing Buffer, and thenincubated for 3 hours at 4° C. with 100 μl/well of alkalinephosphatase-conjugated anti-mouse polyclonal antibody, IgA+IgG+IgM(Zymed, South San Francisco, Calif.) diluted 1:2000 in Diluting Buffer.The plates were then washed 5 times with Washing Buffer, and 100 μl offreshly prepared substrate (p-nitrophenyl phosphate, Sigma, St. Louis,Mo.) diluted to 1 mg/ml in Substrate Buffer was added to each well.Absorbance values were measured after 30 minutes at 405 nm.

Representative binding data to the tethered Pep 4 and Pep 8 are shown inFIGS. 8-A and 8-B, respectively. Several of the SEAM anti-NPr-MenB PSmonoclonal antibodies recognize these two peptides. In contrast,irrelevant mouse monoclonal antibodies of the same isotypes show nobinding in this assay (data not shown). For some of the SEAManti-NPr-MenB PS monoclonal antibodies, the addition of NPr-MenB PS at25 μg/ml completely inhibited binding of the antibody to the peptides(e.g., SEAM-3). For other antibodies, there is either partial inhibitionof binding (e.g., SEAM-16 and SEAM-18), or no inhibition (SEAM-5). Assummarized in Table 5, there is a close correspondence between theconcentration-dependent binding of the SEAM anti-NPr-MenB PS monoclonalantibodies to NPr-MenB PS and the respective binding to particularsynthetic peptides. See, for example, the relative binding of antibodiesSEAM-3, SEAM-5, SEAM-7, SEAM-16, and SEAM-18 to NPr-MenB PS and to Pep8.

TABLE 5 Relative Binding of SEAM Monoclonal Antibodies To NPr- ToSynthetic Lauryl-GLY-GLY-Peptides^(b) MenB PS^(a) Pep 1^(c) Pep 2 Pep 3Pep 4 Pep 6 Pep 7 Pep 8 Pep 9 SEAM-3 0.004 — — 0.016 0.014 — — 0.0090.019 SEAM-5 5 — 47 3 3 — — 3 23 SEAM-7 15 81 80 11 6 25 — 11 60 SEAM-0.08 0.2 — — 0.2 — — 0.06 — 16 SEAM- 0.14 0.8 — 0.8 0.4  1 — 0.2 — 18^(a)In μg/ml of monoclonal antibody, (—) indicates no detectable bindingin the ELISA. ^(b)Concentration of monoclonal antibody required to givean OD of 0.5 at 405 nm after 30 min. incubation with substrate in ELISA.^(c)See FIG. 7 for the amino acid sequences of peptides Pep 1–Pep 4 andPep 6–Pep 9.

EXAMPLE 8 Preparation of Peptide Mimetic Vaccine Compositions

Vaccine compositions containing synthetic peptides corresponding to theabove-described peptide mimetic sequences were prepared as follows.

Preparation of OMP Vesicles. OMP vesicles were prepared from thecapsular-deficient mutant strain of Neisseria meningitidis Group B(Strain M7), using a combination of the techniques described by Lowellet al. (1988) J. Expt. Med. 167:658–663 and Zollinger et al. (1979) J.Clin. Invest. 63:836–848. In brief, Neisseria meningitidis strain M7 (anoncapsular mutant strain derived from NmB), from an overnight cultureon chocolate agar plates incubated at 37° C., was used to inoculate two500 ml flasks of sterile Frantz medium (10.3 g of Na₂HPO₄, 10 g ofcasamino acids (Difco, Detroit, Mich.), 0.36 g of KCl, 0.012 ofcysteine-HCl (SIGMA, St. Louis, Mo.), and 25 ml of 40% glucose-40 mMMgSO₄ (SIGMA, St. Louis, Mo.) in 1 L of water, pH 7.4). The bacteriawere grown from an initial OD of 0.1–0.2 to log phase (OD of 0.75–0.85)on a shaker at 180 rpm for 6–8 hours. The bacteria were inactivated with0.5% phenol solution for one hour at room temperature. The cells wereharvested by centrifuging for 30 minutes at 3000×g. The supernatant wasdecanted, and the cells were washed twice with PBS. The resultant pelletwas stored at −20° C.

The bacteria were then resuspended in 15 ml buffer containing 0.05 MTris-HCl, 0.15 M NaCl and 0.01M EDTA (pH 7.4), and then warmed to 56° C.for 30 minutes. After cooling to room temperature, the suspension wassheared in a POLYTRON (Kinematica GmbH., Luzern, Switzerland) at fullspeed for 3 minutes and then centrifuged at 16000×g for 15 minutes. Theresulting pellet was resuspended with 10 ml buffer (500 mM sodiumchloride, 50 mM sodium phosphate), and treated with 5 ml of DetergentSolution (10% sodium deoxycholate (DOC) (Calbiochem, La Jolla, Calif.),0.15 M glycine (Biorad, Hercules, Calif.) and 30 mMethylenediaminetetraacetic acid (EDTA) (SIGMA, Saint Louis, Mo.). Thesuspension was centrifuged at 16,000×g for 15 minutes. The supernatantwas then collected and centrifuged at 100,000×g for 2 hrs. A pelletcontaining the outer membrane protein preparation was resuspended in 10ml of water and stored at 4° C.

The 10 ml suspension of outer membrane protein was retreated with 5 mlof the Detergent Solution, and then warmed to 56EC for 30 minutes. Aftercooling, lipopolysaccharide (LPS) was removed from the outer membraneprotein by chromatography, 2 ml at a time, using a 2 cm×20 cm SEPHADEXG-100 column (Pharmacia Fine Chemicals, Piscataway, N.J.) in a seconddetergent solution (1% DOC, 0.05 M glycine, and 0.005 M EDTA, pH 8.8).The peak fractions were collected, warmed to 30EC and sterile-filteredthrough a 0.2 μm membrane filter directly into 4 volumes of cold,filter-sterilized ethanol. This mixture was incubated at 4EC overnight.The resulting precipitate was collected by centrifugation at 16,000×gfor 10 minutes, and resuspended in 1 ml of sterile distilled water. Theresulting OMP preparation was soluble but slightly opalescent, and wasstored at −60EC.

Preparation of Peptide/OMP Vesicles. Vaccines were prepared frompeptides Pep 5 and Pep 8, or from a mixture of peptides Pep 1–Pep 9. Tofacilitate hydrophobic complexing of the peptides to the OMP vesicle,each peptide was modified by the addition at the amino terminus of ahydrophobic tail (Lauryl-GLY-GLY) and a carboxyl amide as describedabove for the ELISA. For each vaccine, 5 mg of peptide was dissolved in100 μl dimethylsulfoxide (DMSO) (SIGMA, Saint Louis, Mo.). The resultingsolution was diluted to 750 μl in buffer containing 50 mM4-(-2-hydroxyethyl)-1-piperazineethanesulfonic Acid (Hepes), pH 8.0, and1 M potassium ferricyanide (SIGMA, Saint Louis, Mo.). 7.5 μg ofzwitterionic detergent (Empigen, Calbiochem, La Jolla, Calif.) was thenadded to the above peptide solution. After incubation at roomtemperature for 1 hour, each of the peptide solutions was combined with250 μl of outer membrane protein (OMP) vesicles (20 mg/ml) for a totalvolume of 1 ml. The solution was heated to 75EC for 20 minutes. Aftercooling to room temperature, the OMP/Peptide mixture was added to aSLIDE-A-LYZER (Pierce, Rockford, Ill.) a dialysis cassette with a 10,000molecular weight cut off, and dialyzed in 1 L PBS overnight. The PBSsolution (1 L) was changed twice over 8 hours.

EXAMPLE 9 Immunization with OMP-Peptide Mimetic Vaccine Compositions

In order to assess the OMP-peptide vaccine compositions prepared inExample 8 above, the following study was carried out.

Animals: Balb/c and CD1 mice (Jackson Laboratory, Bar Harbor, Me.) wereused for the immunogenicity studies. Mice were kept in quarantine for 2weeks.

Vaccine Preparations: For the first injection, vaccine solutions (2mg/ml total peptide/protein in PBS) were combined with equal volumes ofcomplete Freund's adjuvant. (Sigma, St. Louis, Mo.) to yield a finalconcentration of 1 mg/ml of peptide/protein. For the subsequentinjections, similar vaccine compositions were prepared using incompleteFreunds adjuvant. The respective compositions were forced back and forththrough 2 syringes in order to obtain homogenous emulsions which werethen used in the immunizations.

Immunizations: Each treatment group included 4 Balb/c mice and 4 CD1mice. There were also control groups of 4 Balb/c and 4 CD1 mice thatwere not immunized. Individual treatment groups received doses of 5 μgor 50 μg of peptide, and 5 μg or 50 μg of OMP Vesicles, respectively.The vaccine composition was administered intraperitonealy (IP), in atotal volume of 5 or 50 μl, respectively. Immunizations were repeated at3 week intervals for a total of 3 immunizations. The animals were bledfrom the tail vein 1 and 4 weeks after the third immunization.

CD1 and Balb/c mice immunized with peptide Pep 8 complexed with OMPvesicles develop high anti-Pep 8 antibody responses as measured by ELISAin serum obtained 4 weeks post-third immunization. Representative datafor the responses of the CD1 mice are shown in FIG. 9. Antibody bindingto tethered Pep 8 is inhibited by soluble Pep 8 (Acetyl-[Pep 8]-Amide)but not by a soluble irrelevant peptide “R1”(Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO. 68)). Anti-Pep 8antibodies also were elicited in mice immunized with a combination ofnine peptides (peptides Pep 1–Pep 9/OMP), but not in mice immunized withPep 5/OMP alone. This demonstrates the Pep 8-specific antibodies wereelicited by Pep 8-containing immunogens.

FIG. 10 summarizes the cross-reactivity of the CD1 mouse immune serawith NPr-MenB PS or NAc-MenB PS in an ELISA assay. All three immunogens(Pep 5/OMP, Pep 8/OMP, and peptides Pep 1–Pep 9/OMP) appeared to elicitserum antibodies cross-reactive with NPr-MenB PS, which were notdetected in the serum pool from the unimmunized control mice. However,the specificity of this antibody binding could not be confirmed sincethere was no significant inhibition observed in wells containing solubleNPr-MenB PS (data not shown). The ability of soluble Pep 8 (Acetyl-[Pep8]-Amide) to inhibit binding of the anti-Pep 8 serum pools to the solidphase NPr-MenB PS also could not be verified since the presence of thispeptide resulted in significant increase in antibody binding which wasnot detected in the presence of a soluble irrelevant peptide “R1”(Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO. 68)).

Data from characterization of the extensive collection of SEAMmonoclonal antibodies indicate that the ability of an antibody to bindto NAc-MenB PS in an ELISA correlates with the presence of autoantibodyactivity as assessed by binding to PSA expressed by CHP-134neuroblastoma cells (see Table 1). FIG. 11 summarizes thecross-reactivity of the CD1 mouse immune sera with NAc-MenB PS in anELISA. None of the serum pooled from the peptide-vaccinated mice werepositive in this assay. In contrast, a SEAM anti-NPr-MenB PS monoclonalantibody with known autoantibody activity was strongly positive in thisassay when tested at 2.0 μg/ml. The lack of cross-reactivity of theanti-Pep antisera with NAc-MenB PS by ELISA indicates that theseantibodies do not have PSA-specific autoantibody activity.

Complement-mediated bactericidal activity of pooled CD1 sera from miceimmunized with either 5 μg or 50 μg of Pep 8/OMP vaccine is shown inFIGS. 12A and 12B, respectively. At both doses, the Pep 8-containingvaccine elicited serum antibodies that were able to mediatebacteriolysis of MenB strain 8047 in the presence of human complement. Aportion of this antibody may have been elicited by the OMP vesicles usedas an adjuvant. However, at serum dilutions of 1:1000, 50% or greater ofthe bactericidal activity was mediated by the anti-Pep 8 antibodies asdemonstrated by inhibition of the reaction with Lauryl-GLY-GLY-Pep 8 ata final serum concentration of 100 μg/ml.

Thus, novel MenB PS antibodies, molecular peptide mimetics capable ofeliciting bactericidal MenB antibody, and method for obtaining and usingthe same are disclosed. Although preferred embodiments of the subjectinvention have been described in some detail, it is understood thatobvious variations can be made without departing from the spirit and thescope of the invention as defined by the appended claims.

Deposits of Strains Useful in Practicing the Invention

Deposits of biologically pure cultures of the following hybridoma celllines were made with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209. The accession numbersindicated were assigned after successful viability testing, and therequisite fees were paid. The deposits were made under the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and the Regulationsthereunder (Budapest Treaty). This assures maintenance of viablecultures for a period of thirty (30) years from the date of deposit. Theorganisms will be made available by the ATCC under the terms of theBudapest Treaty, and subject to an agreement between Chiron Corporationand the ATCC, which assures permanent and unrestricted availability ofthe progeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 U.S.C. §122 and theCommissioner's rules pursuant thereto (including 37 C.F.R. §1.12 withparticular reference to 886 OG 638). Upon the granting of a patent, allrestrictions on the availability to the public of the deposited cultureswill be irrevocably removed.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The nucleic acid sequences of these hybridomas, as well asthe amino acid sequences of the antibody molecules encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with the description herein. A license may be required to make,use, or sell the deposited materials, and no such license is herebygranted.

HYBRIDOMA Deposit Date ATCC No. SEAM-3 Aug. 16, 1996 HB-12170 SEAM-18Aug. 16, 1996 HB-12169 SEAM-2 Jul. 30, 1997 CRL-12380 SEAM-12 Jul. 30,1997 CRL-12381

1. A method for identifying a molecular mimetic of a unique epitope ofNeisseria meningitidis serogroup B (MenB), said method comprising: (a)providing a library of molecules comprising a putative molecular mimeticof a unique epitope of MenB, wherein said library of molecules isselected from the group consisting of a peptoid library, a peptidelibrary and a phage-display library; (b) contacting said library ofmolecules with an isolated antibody immunologically reactive with anN-acyl-substituted Neisseria meningitidis serogroup B capsularpolysaccharide, wherein said antibody is not autoreactive with Neisseriameningitidis serogroup B capsular polysaccharide as determined bymeasuring the ability of said antibody to react with human neuroblastomacell line CHP-134, under conditions that allow immunological bindingbetween said antibody and said molecular mimetic, if present, to providea complex; and (c) separating the complex from non-bound molecules; and(d) identifying the molecular mimetic present in the complex.
 2. Themethod of claim 1 wherein said library of molecules is a peptoidlibrary.
 3. The method of claim 1 wherein said library of molecules is apeptide library.
 4. The method of claim 1 wherein said library ofmolecules is phage-display library.